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An Improved Method to Determine Cell Viability by Simultaneous Staining with Fluorescein Diacetate–Propidium Iodide
Abstract
A rapid, simultaneous double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) is described for use in the determination of cell viability in cell suspension. Air-dried slide preparations can be made from the cell suspensions so that an accurate estimate of the viability of the cells in the original suspension can be made up to 1 week later. Viable cells fluoresce bright green, while nonviable cells are bright red. Furthermore, when FDA-PI staining is compared to trypan blue dye exclusion as a method to determine cell viability, FDA-PI is found to be more consistent over prolonged periods of exposure to the dyes. Therefore, double staining with FDA-PI is a rapid, convenient, and reliable method to determine cell viability.
... Trypan blue exclusion is the most common test for cell viability; however, a number of limitations of this assay are related to the exclusion of certain dyes by live cell membranes. For example, cells must be counted within 3-5 min from the dye staining, because the number of blue-staining cells increases with time after the dye's addition [74]. When counting a large number of samples, it is extremely inconvenient to perform all the tests on the same day by counting one cell suspension at a time before staining the next sample. ...
... In this study, three different types of assays were performed to gain clues about the mode of cell death induced by H 2 O 2 against H400, H357, and OKF6 cells: trypan blue exclusion, phase contrast microscopy, and fluorescence-based live-dead assay. Trypan blue exclusion is the most common test that can exclude live cells from non-viable cells [74]. Trypan blue exclusion study showed that H 2 O 2 induced cell deathin H400 OSCC cells through a shift in live cell population proportional to increasing H 2 O 2 concentrations from 2 to 24 h. ...
Reactive oxygen species (ROS) are highly reactive molecules generated in living organisms and an excessive production of ROS culminates in oxidative stress and cellular damage. Notably, oxidative stress plays a critical role in the pathogenesis of a number of oral mucosal diseases, including oral mucositis, which remains one of cancer treatments’ most common side effects. We have shown previously that oral keratinocytes are remarkably sensitive to oxidative stress, and this may hinder the development and reproducibility of epithelial cell-based models of oral disease. Here, we examined the oxidative stress signatures that parallel oral toxicity by reproducing the initial events taking place during cancer treatment-induced oral mucositis. We used three oral epithelial cell lines (an immortalized normal human oral keratinocyte cell line, OKF6, and malignant oral keratinocytes, H357 and H400), as well as a mouse model of mucositis. The cells were subjected to increasing oxidative stress by incubation with hydrogen peroxide (H2O2) at concentrations of 100 μM up to 1200 μM, for up to 24 h, and ROS production and real-time kinetics of oxidative stress were investigated using fluorescent dye-based probes. Cell viability was assessed using a trypan blue exclusion assay, a fluorescence-based live–dead assay, and a fluorometric cytotoxicity assay (FCA), while morphological changes were analyzed by means of a phase-contrast inverted microscope. Static and dynamic real-time detection of the redox changes in keratinocytes showed a time-dependent increase of ROS production during oxidative stress-induced epithelial injury. The survival rates of oral epithelial cells were significantly affected after exposure to oxidative stress in a dose- and cell line-dependent manner. Values of TC50 of 800 μM, 800 μM, and 400 μM were reported for H400 cells (54.21 ± 9.04, p < 0.01), H357 cells (53.48 ± 4.01, p < 0.01), and OKF6 cells (48.64 ± 3.09, p < 0.01), respectively. Oxidative stress markers (MPO and MDA) were also significantly increased in oral tissues in our dual mouse model of chemotherapy-induced mucositis. In summary, we characterized and validated an oxidative stress model in human oral keratinocytes and identified optimal experimental conditions for the study of oxidative stress-induced oral epithelial toxicity.
... Instead, Cu6-PLGA NPs were not able to enter cells because the latter were dead. It is known that propidium iodide is a membrane-impermeable dye that is generally excluded from viable cells and, conversely, fills the cytoplasm with non-viable cells [24]. Interestingly, the cytotoxicity test performed on roots treated with 80 µM dynasore for 120 min showed that the roots were viable (Figure 4b) and, after Cu6-PLGA NPs treatment, fluorescence was clearly visible in the epidermis (Figure 4a). ...
... The uptake of NPs in plants is species-specific and depends on the NPs' properties, such as size, porosity, hydrophobicity, and surface area. For example, it is known that Au NPs are taken up by tobacco but not by wheat [20][21][22][23][24][25]. The porous nature of cell walls allows NPs to cross them by binding to protein carriers via aquaporins, ion channels, endocytosis, or by piercing the cell membrane and creating new pores [26][27][28]. ...
To date, most endocytosis studies in plant cells have focused on clathrin-dependent endocytosis, while limited evidence is available on clathrin-independent pathways. Since dynamin a is a key protein both in clathrin-mediated endocytosis and in clathrin-independent endocytic processes, this study investigated its role in the uptake of poly-(lactic-co-glycolic) acid (PLGA) nanoparticles (NPs). The experiments were performed on cultured cells and roots of Arabidopsis thaliana. Dynasore was used to inhibit the activity of dynamin-like proteins to investigate whether PLGA NPs enter plant cells through a dynamin-like-dependent or dynamin-like-independent endocytic pathway. Observations were performed by confocal microscopy using a fluorescent probe, coumarin 6, loaded in PLGA NPs. The results showed that both cells and roots of A. thaliana rapidly take up PLGA NPs. Dynasore was administered at different concentrations and exposure times in order to identify the effective ones for inhibitory activity. Treatments with dynasore did not prevent the NPs uptake, as revealed by the presence of fluorescence emission detected in the cytoplasm. At the highest concentration and the longest exposure time to dynasore, the fluorescence of NPs was not visible due to cell death. Thus, the results suggest that, because the NPs’ uptake is unaffected by dynasore exposure, NPs can enter cells and roots by following a dynamin-like-independent endocytic pathway.
... DMSO was used to solubilize the crystals prior to colorimetric measurement at 590 nm wavelength using a microplate reader (InfiniteM200pro, Switzerland). Sf9 cell lines treated with lutein and untreated cells were centrifuged at 200 × g and the pellet containing cells were treated with the fluorescent dyes fluorescein diacetate (FDA) and propidium iodide (P.I) [30]. Cell viability and morphology were observed under Eclipse Ti-S phase contrast fluorescence inverted microscope (Nikon, USA). ...
... Fluorescent microscopic images of the cell line shown in Fig. 8 0.9 µg/mL emitted green fluorescent light. Maximum red fluoresce was observed in cells treated with 6 µg/mL or higher lutein concentration which indicates the death or damage to the cells [30]. At high lutein concentration, cells were irregular in shape with reduced size, and cell debris was observed due to cellular damage. ...
Production of value-added products from waste anaerobic digestate is economically and environmentally important for sustainable development of industrial process and products. In this study halophilic microalgae, Chlorella vulgaris 92001, Chlorella vulgaris 50291, Chlorella vulgaris 10241 and Tetraselmis indica, were initially screened for lutein production using synthetic dairy digestate (DD), municipal digestate (MD) and poultry digestate (PD) as no-cost substrates. Screening and optimization of parameters, such as dilution, pH, MgCl2, NaCl, NaHCO3 and inoculum concentration for maximum lutein production were further performed employing statistically designed Plackett–Burman and response surface methodology. Cultivation of C. vulgaris 92001 in a split column photobioreactor under optimum culture condition showed increase in lutein production by 2.36-fold in batch mode. The influence of different hydraulic retention time (HRT) values of 150, 130, 100 and 90 h on lutein production was evaluated in continuous mode with the split column photobioreactor. Lutein produced using the synthetic poultry digestate showed good potential biopesticide activity against Spodoptera litura (fall armyworm). Overall, this study demonstrated bioprocess development to produce lutein using synthetic anaerobic digestate from marine algae and its potential application as a biopesticide.
... Isaria cateniannulata (Liang) Sanmson & Hywel-Jones (Hypocreales: Clavicipitaceae), also known as Paecilomyces cateniannulatus or Cordyceps cateniannulata [12][13][14], is an important pathogenic fungus of insects that has been utilized for the biocontrol of agricultural and forestry pests [15][16][17][18][19][20][21]. Recently, some scholars have found that it can promote the growth of buckwheat, tomato, tobacco, and so on [22][23][24][25], but the method of to colonizing plants and promoting plant growth has not been explored yet. ...
... A working solution was freshly prepared for each experiment by adding 35 µL of FDA liquid to 4 mL of deionized water. Samples were maintained on ice, protected from light [15] and were stored at 4 • C for up to 6 months protected from light [25]. ...
The use of entomogenous fungi as endophytes is currently an area of active research. Isaria cateniannulata is an important entomogenous fungus that has been employed for the active control of a range of pests in agricultural and forestry settings, but its direct impact on plants remains to be evaluated. Herein, we assessed the ability of I. cateniannulata to colonize buckwheat, Fagopyrum esculentum and F. tataricum, and its impact on buckwheat defense enzyme activity and physiological indexes. The majority of fungal submerge condia was able to enter into leaves through stomata and veins, and this was followed by conidial attachment, lytic enzyme secretion, conidial deformation, and enhanced defensive enzyme activity within buckwheat, followed by the repair of damaged tissue structures. I. cateniannulata populations on buckwheat leaf surfaces (in CFU/g) reached the minimum values at 24 h after inoculation. At this time, the blast analysis revealed that the sequence identity values were 100%, which was consistent with the sequence of I. cateniannula. The number of I. cateniannulata submerge conidia colonized in the buckwheat leaves gradually rose to peak levels on 7 d post-inoculation, and then gradually declined until 10 d, at which time the buckwheat plant growth index values increased. This study provided novel evidence that I. cateniannulata could be leveraged as an endophytic fungus capable of colonizing buckwheat plants and promoting their growth.
... Regarding trypan blue staining, while it may be less suitable for adherent cells due to its requirement for cell suspension, it remains a viable alternative for staining suspension cells, particularly when PI/FDA staining is not feasible. One should keep in mind that when PI/FDA staining is compared to trypan blue dye exclusion as a method to determine cell viability, PI/FDA is found to be more consistent over prolonged periods of exposure to the dyes [26]. This versatility underscores the importance of selecting the most appropriate staining method based on the specific characteristics and requirements of the experimental setup. ...
Tissue engineering holds promise for developing therapeutic applications using viscous materials e.g. hydrogels. However, assessing the cytotoxicity of such materials with conventional assays can be challenging due to non-specific interactions. To address this, we optimized a live/dead staining method for quantitative evaluation and compared it with the conventional CCK8 assay. Our MicroDrop method involved seeding droplets containing 5000 cells in 10 µl medium on 12-well plates. After allowing them to adhere for 4 h, various viscous samples were applied to the cells and measurements were conducted using a fluorescence microscope immediately and at daily intervals up to 72 h. A sodium dodecyl sulfate (SDS) dilution series compared the MicroDrop with the CCK8 assay. The findings revealed a cell-type specific pattern for 10 mg/ml hyaluronic acid (HA), wherein MC3T3-E1 cells maintained 95% viability until 72 h, while L929 cells experienced a gradual decline to 17%. 2 mg/ml HA exhibited consistent viability above 90% across all time points and cell lines. Similarly, fibrin demonstrated 90% viability across dilutions and time points, except for undiluted samples showing a decrease from 85% to 20%. Gelatin-methacrylol sustained viability above 70% across all time points at both 5% and 10% concentrations. The comparison of the SDS dilution series between viability (MicroDrop) and metabolic activity (CCK8) assay showed a correlation coefficient of 0.95. The study validates the feasibility of the established assay, providing researchers with an efficient tool for assessing cytotoxicity in viscous materials. Additionally, it holds the potential to yield more precise data on well-known hydrogels.
Graphical Abstract
... Fluorescein diacetate (FDA) is a substrate for cell-permeant esterase that can be used as a viability test indicator according to the method of Jones and Senft [52]. After 24 hours of treatment with various percentages of DMSO (0.041 to 1.33%) and ethanol (0.0325 to 1.04%), and different concentrations of oxysterols (15. ) production was measured by flow cytometry after staining with dihydroethidium (DHE) according to the method of Rothe and Valet [53]. ...
Milk thistle seed oil is still not a well-known edible oil. Silybum marianum (milk thistle), is present in several countries and is the only known representative of the genus Silybum. However, Silybum eburneum, which is an endemic plant in Spain, Kenya, Morocco, Algeria, and Tunisia, is considered a marginalized species. The present work is the first report that gives information on the lipid and phenolic profiles of Tunisian S. eburneum seed oil compared to those of Tunisian S. marianum seed oil. In addition, the antioxidant properties of these oils were determined with DPPH, FRAP, and KRL assays, and their ability to prevent oxidative stress was determined on human monocytic THP-1 cells. These oils are characterized by high amounts of unsaturated fatty acids; linoleic acid and oleic acid are the most abundant. Campesterol, sitosterol, stigmasterol, and β-amyrin were the major phytosterols identified. α-tocopherol was the predominant tocopherol found. These oils also contain significant amounts of phenolic compounds. The diversity and richness of Silybum marianum and Silybum eburneum seed oils in unsaturated fatty acids, phenolic compounds, and tocopherols are associated with high antioxidant activities revealed by the DPPH, FRAP, and KRL assays. In addition, on THP-1 cells, these oils powerfully reduced the oxidative stress induced by 7-ketocholesterol and 7β-hydroxycholesterol, two strongly pro-oxidant oxysterols often present at increased levels in patients with age-related diseases. Silybum marianum and Silybum eburneum seed oils are therefore important sources of bioactive molecules with nutritional interest that prevent age-related diseases, the frequency of which is increasing in all countries due to the length of life expectancy.
... The percentage viability of each sample was determined based on the number of cells that were stained green divided by the total number of cells counted. For both cell morphology and viability, observations were made using an epifluorescence microscope [64]. Objectives with 40× and 10× magnification were used. ...
Bursera fagaroides, popularly used in México, possesses bioactive lignans. These compounds are low in the bark, and its extraction endangers the life of the trees. The aim of the present investigation was to search for alternative sources of cytotoxic compounds in B. fagaroides prepared as leaves and in vitro callus cultures. The friable callus of B. fagaroides was established using a combination of plant growth regulators: 4 mgL−1 of 2,4-dichlorophenoxyacetic acid (2,4-D), 1 mgL−1 Naphthaleneacetic Acid (NAA) and 1 mgL−1 Zeatin. The maximum cell growth was at day 28 with a specific growth rate of μ = 0.059 days−1 and duplication time td = 11.8 days. HPLC quantification of the dichloromethane callus biomass extract showed that Scopoletin, with a concentration of 10.7 µg g−1 dry weight, was the main compound inducible as a phytoalexin by the addition of high concentrations of 2,4-D, as well as by the absence of nutrients in the culture medium. In this same extract, the compounds γ-sitosterol and stigmasterol were also identified by GC-MS analysis. Open column chromatography was used to separate and identify yatein, acetyl podophyllotoxin and 7′,8′-dehydropodophyllotoxin in the leaves of the wild plant. Cytotoxic activity on four cancer cell lines was tested, with PC-3 prostate carcinoma (IC50 of 12.6 ± 4.6 µgmL−1) being the most sensitive to the wild-type plant extract and HeLa cervical carcinoma (IC50 of 72 ± 5 µgmL−1) being the most sensitive to the callus culture extract.
... The total chlorophyll content and chlorophyll a, b were determined by measuring absorbance values at 663 and 646 nm with an ultraviolet spectrophotometer (Shimadzu). Cell viability was determined (Jones and Senft 1985) by treating the lettuce root tips in a dye solution FDA-PI containing 12.5 g mL À1 Fluorescein diacetate (FDA) and 5 g mL À1 Propidium iodide (PI), and darkened for 10 min, In the formula: C a , C b , and C T are the concentrations of chlorophyll a, chlorophyll b, and total chlorophyll, respectively; A and A6 represents the absorbance measurements of the extract at 663 and 646 nm, respectively; C is the concentration of chloroplast pigment, and V is the extraction liquid volume. ...
The ancient bayberry demonstrates superior resistance to both biotic and abiotic stresses compared to cultivated bayberry, yet the underlying mechanisms remain largely unexplored. This study investigates whether long‐term bayberry cultivation enhances stress resistance through modulation of tissue‐specific microbes and metabolites. Employing microbiome amplicon sequencing alongside untargeted mass spectrometry analysis, we scrutinize the role of endosphere and rhizosphere microbial communities and metabolites in shaping the differential resistance observed between ancient and cultivated bayberry trees. Our findings highlight the presence of core microbiome and metabolites across various bayberry tissues, suggesting that the heightened resistance of ancient bayberry may stem from alterations in rhizosphere and endosphere microbial communities and secondary metabolites. Specifically, enrichment of Bacillus in roots and stems, Pseudomonas in leaves, and Mortierella in rhizosphere soil of ancient bayberry was noted. Furthermore, correlation analysis underscores the significance of enriched microbial species in enhancing ancient bayberry's resistance to stresses, with elevated levels of resistance‐associated metabolites such as beta‐myrcene, benzothiazole, L‐glutamic acid, and gamma‐aminobutyric acid identified through GC–MS metabolomics analysis. The beneficial role of these resistance‐associated metabolites was further elucidated through assessment of their promotive and allelopathic effects, as well as their phytostatic and antioxidant functions in lettuce plants. Ultimately, our study delves into the intrinsic reasons behind the greater resistance of ancient bayberry to biotic and abiotic stresses by evaluating the impact of long‐term planting on the microbial community and metabolites in the bayberry endosphere and rhizosphere, shedding light on the complex dynamics of host‐microbial interactions.
... High-performance liquid chromatography (HPLC) equipped with a dual pump with micro-vacuum degasser and UV detector (UV) (Prostar 210, Varian, USA) was used to measure CPS concentration at 230 nm wavelength using analytical column (Agilent 5 TC-C18, particle size 5 μm, 250 × 4.5 mm) following the procedure suggested by Liu et al. (2016). A previous study by Kumar et al., 2023a presented methodology for the estimation of interface settling velocity (ISV) (Metcalf and Eddy, 2003), aerobic sludge biomass activity (ASBA) (Negi et al., 2020(Negi et al., , 2022, sludge volume index (SVI) (APHA et al., 2005), filamentous index (FI) (Eikelboom, 2000), microscopic observation, photographic evidence, cell viability test (Jones and Senft, 1985), extracellular polymeric substance (EPS) (Tomar and Chakraborty, 2018), polysaccharides (PS) (Frølund et al., 1996), Protein (PN) (Lowry et al., 1951), enzymatic activities (catalase, dehydrogenase, protease, and urease) (Lv et al., 2008;Yao et al., 2010) and amplified ribosomal DNA restriction analysis (ARDRA) (Gich et al., 2000;Meng et al., 2015). ...
Non-agricultural sources of pesticides in urban areas are responsible for their presence in domestic wastewater. Therefore, pesticides are typically found in sewage treatment plants in developed and developing countries as micro-pollutant. The presence of pesticides in the wastewater can impart stress on the aerobic sludge biomass and disrupt the functioning of the plant. However, there exists a knowledge gap regarding the resilience of aerobic sludge biomass towards stress due to the presence of pesticides in the wastewater. This study investigated the impact of chlorpyrifos (CPS) – a widely used pesticide, on sludge biomass and explored its recovery capability when CPS is discontinued in the influent. Four duplicate reactors were operated with different CPS concentrations ranging from 50 – 200 mg/L. Chemical oxygen demand (COD) removal for reactors has ranged within 18-73% at the steady state of the stressed phase, whereas COD removal for the control reactor was 91%. CPS stress slightly inhibited filamentous biomass growth. Biomass activity and cell viability have decreased significantly, whereas biochemical contents have varied slightly under CPS stress. The activities of the enzymes dehydrogenase and urease were significantly inhibited when compared to catalase and protease. Amplified ribosomal DNA restriction analysis reflected changes in the microbial community. The discontinuation of CPS has allowed aerobic sludge biomass to recover in its organic degradation capability (COD removal of more than 88% at steady-state conditions of recovery phase operation), biomass growth, and cell viability. In addition, enzyme activities have retrieved to their original levels, and 78-93% similarity of microbial community structure has been displayed between CPS-exposed and control reactor biomasses. Overall, the present study has indicated the orderly changes in the quality of aerobic sludge biomass under CPS stress through physico-chemical and biological characteristics. The study also has highlighted the self-recovery of sludge biomass characteristics stressed with different concentrations of CPS.
... In order to further study the toxicity of tauMBD to PC12 cell lines, the fluorescein diacetate/propidium iodide (FDA/ PI) mixed double staining method were also performed to distinguish live and dead cells [26]. The cells were seeded into 6-well plates with 2 mL fresh medium to a density of ∼ 5 × 10 4 cells per well. ...
Background
Neurofibrillary tangles (NFTs) are one of the most common pathological characteristics of Alzheimer’s disease. The NFTs are mainly composed of hyperphosphorylated microtubule-associated tau. Thus, recombinant tau is urgently required for the study of its fibrillogenesis and its associated cytotoxicity.
Methods and results
Heterologous expression, purification, and fibrillation of the microtubule-binding domain (MBD) of tau (tauMBD) were performed. The tauMBD was heterologously expressed in E. coli. Ni-chelating affinity chromatography was then performed to purify the target protein. Thereafter, tauMBD was systematically identified using the SDS-PAGE, western blot and MALDI-TOF MS methods. The aggregation propensity of the tauMBD was explored by both the thioflavin T fluorescence and atomic force microscopy experiments.
Conclusions
The final yield of the recombinant tauMBD was ~ 20 mg L⁻¹. It is shown that TauMBD, in the absence of an inducer, self-assembled into the typical fibrils at a faster rate than wild-type tau. Finally, the in vitro cytotoxicity of tauMBD aggregates was validated using PC12 cells. The heterologously expressed tau in this study can be further used in the investigation of the biophysical and cellular cytotoxic properties of tau.
Graphical abstract
... Cell counters can determine whether fluorescently stained cells are alive or dead. Cell death can be roughly divided into two types: apoptosis and necrosis, but Cell counters cannot distinguish apoptosis from necrosis in dead cells [19], [20], [21], [22]. Distinguishing between apoptosis and necrosis is very important in determining whether an approach to cells is effective. ...
Cell counters, which are dedicated cell analyzers, can be used to analyze cellular status. Cell counters are smaller and less expensive (about $13,000) than other cell analysis devices such as flow cytometers (FACS), real-time PCR, and sequencers, and can discriminate between life and death of fluorescently stained cells. Cell death can be roughly divided into two types: apoptosis and necrosis, but Cell counters cannot distinguish between apoptosis and necrosis in cells. This study developed a biochip system for inexpensive, simple, and capable of distinguishing between live, apoptotic, and necrotic cells. This biochip system (70 x 150 x 80 mm) comprises a slide into which fluorescently stained cells are injected, an LED light source, and a camera system. When cells stained with a fluorescent reagent are irradiated at the excitation wavelength, they fluoresce. By changing the combination of fluorescent reagent and excitation wavelength, live, apoptotic, and necrotic cells can be photographed. Then they are processed by a cell counting program using existing methods to determine numbers of live, dead, and necrotic cells. To demonstrate the effectiveness of this system, we conducted live cell, apoptosis, and necrosis detection experiments using colon cancer cells. Results of each experiment using the biochip system were compared with visual cell counts made by an operator. The novel biochip system successfully distinguishes between live, apoptotic and necrotic cells. Detection time was <1 s, and the detection error was 9%, compared to visual inspection.
... For viability assays, one ovarian tissue fragment from each female was treated with FDA and PI [21]. ...
This study aimed to evaluate the viability of reproductive gametes in zebrafish ( Danio rerio ), at different rigor mortis stages. Viability assessments were conducted on oocytes at various developmental stages using LIVE/DEAD and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. For sperm evaluation, both kinetic (CASA) and morphological assessments (Rose Bengal staining) were performed. Results demonstrated that rigor mortis progression significantly impacted oocyte viability during post-rigor stages, with the following viability rates: pre- rigor (70.43 ± 12.31%), fresh/control (46.43 ± 12.54%), post-rigor (27.62 ± 22.29%), and rigor mortis (comparable to fresh/control). Conversely, sperm kinetics exhibited nuanced responses to the rigor mortis stages, with specific parameters showing sensitivity, whereas the others remained relatively stable. Sperm motility was higher in the fresh/control (63.23 ± 19.03%) and pre- rigor (58.96 ± 14.38%) compared to the post-rigor group (3.34 ± 4.65%). This study highlights the significance of the pre- rigor for successful gamete collection and preservation. These findings provide valuable insights for conservation efforts and optimization of genetic resource management for endangered fish species. This study aimed to develop effective assistive reproductive techniques by elucidating the interplay between rigor mortis and gamete quality, contributing to the broader goals of species conservation and maintenance of genetic diversity in fish populations.
... Conventional C-domains, only capable of condensing Lamino acids (i.e., L C L -domains) into the growing peptide chain and devoid of epimerization functionality, feature an active site with a conserved histidine-containing HHxxxDG motif, in which the second histidine (H) is crucial for catalysis [15,38]. The dual E/C-domains present in Pseudomonas CLiPs carry an equivalent HHxxxDH motif, with an extra histidine (or less frequently, alanine or asparagine) substituting for the glycine [15,39]. In addition, the dual E/C-domains invariably exhibit a second histidine sequence motif, with the proposed consensus sequence HH[I/L]xxxxGD located close to the N-terminus of the domain [23,38]. ...
In Pseudomonas lipopeptides, the D-configuration of amino acids is generated by dedicated, dual-function epimerization/condensation (E/C) domains. The increasing attention to stereochemistry in lipopeptide structure elucidation efforts has revealed multiple examples where epimerization does not occur, even though an E/C-type domain is present. While the origin of the idle epimerization in those E/C-domains remains elusive, epimerization activity has so far shown a binary profile: it is either ‘on’ (active) or ‘off’ (inactive). Here, we report the unprecedented observation of an E/C-domain that acts ‘on and off’, giving rise to the production of two diastereoisomeric lipopeptides by a single non-ribosomal peptide synthetase system. Using dereplication based on solid-phase peptide synthesis and NMR fingerprinting, we first show that the two cyclic lipopeptides produced by Pseudomonas entomophila COR5 correspond to entolysin A and B originally described for P. entomophila L48. Next, we prove that both are diastereoisomeric homologues differing only in the configuration of a single amino acid. This configurational variability is maintained in multiple Pseudomonas strains and typically occurs in a 3:2 ratio. Bioinformatic analysis reveals a possible correlation with the composition of the flanking sequence of the N-terminal secondary histidine motif characteristic for dual-function E/C-type domains. In permeabilization assays, using propidium iodide entolysin B has a higher antifungal activity compared to entolysin A against Botrytis cinerea and Pyricularia oryzae spores. The fact that configurational homologues are produced by the same NRPS system in a Pseudomonas strain adds a new level of structural and functional diversification to those already known from substrate flexibility during the recruitment of the amino acids and fatty acids and underscores the importance of complete stereochemical elucidation of non-ribosomal lipopeptide structures.
... However, in some other studies, it tends to overestimate pollen germination frequency (Wang et al., 2004;Dutta et al., 2013;Impe et al., 2020), particularly when applied to samples containing immature pollen (Heslop-Harrison et al., 1984). As an improvement of FDA staining, FDA-propidium iodide (FDA-PI) (Jones and Senft, 1985) double staining significantly improves the ability to discriminate between live and dead pollen (Dupl'áková et al., 2016;Ascari et al., 2020). Recently, the dichlorodihydrofluorescein diacetate (H 2 DCFDA) staining method was developed utilizing the non-cytotoxic reactive oxygen species (ROS) probe H 2 DCFDA to predict pollen germination by measuring the ROS dynamics of pollen grains, which enables the accurate assessment of the viability of a large number of pollen grains when used in combination with flow cytometry (Luria et al., 2019;Rutley and Miller, 2020). ...
In vitro pollen germination is considered the most efficient method to assess pollen viability. The pollen germination frequency and pollen tube length, which are key indicators of pollen viability, should be accurately measured during in vitro culture. In this study, a Mask R-CNN model trained using microscopic images of tree peony (Paeonia suffruticosa) pollen has been proposed to rapidly detect pollen germination rate and pollen tube length. To reduce the workload during image acquisition, images of synthesized crossed pollen tube were added to the training dataset, significantly improving the model accuracy in recognizing crossed pollen tubes. At an Intersection over Union threshold of 50%, a mean average precision of 0.949 was achieved. The performance of the model was verified using 120 testing images. The R 2 value of the linear regression model using detected pollen germination frequency against the ground truth was 0.909 and that using average pollen tube length was 0.958. Further, the model was successfully applied to two other plant species indicating a good generalizability and potential to be applied widely.
... Widely used assays of plasma membrane deterioration (Segawa and Nagata, 2015;Zhang et al., 2018) are based on the membrane permeability of normally excluded dyes, such as silver, Fluoro-Jade, ethidium bromide, and propidium iodide (PI) (K. H. Jones and Senft, 1985;Switzer, 2000;Bouchier-Hayes et al., 2008;Schmued, 2016). The brain slice preparation is useful for the study of neuronal death after acute injury. ...
In in vitro models of acute brain injury, neuronal death may overwhelm the capacity for microglial phagocytosis, creating a queue of dying neurons awaiting clearance. Neurons undergoing programmed cell death are in this queue, and are the most visible and frequently quantified measure of neuronal death after injury. However, the size of this queue should be equally sensitive to changes in neuronal death and the rate of phagocytosis. Using rodent organotypic hippocampal slice cultures as a model of acute perinatal brain injury, serial imaging demonstrated that the capacity for microglial phagocytosis of dying neurons was overwhelmed for two weeks. Altering phagocytosis rates, e.g. by changing the number of microglia, dramatically changed the number of visibly dying neurons. Similar effects were generated when the visibility of dying neurons was altered by changing the membrane permeability for stains that label dying neurons. Canonically neuroprotective interventions such as seizure blockade and neurotoxic maneuvers such as perinatal ethanol exposure were mediated by effects on microglial activity and the membrane permeability of neurons undergoing programmed cell death. These canonically neuroprotective and neurotoxic interventions had either no or opposing effects on healthy surviving neurons identified by the ongoing expression of transgenic fluorescent proteins.
Significance Statement:
In in vitro models of acute brain injury, microglial phagocytosis is overwhelmed by the number of dying cells. Under these conditions, the assumptions on which assays for neuroprotective and neurotoxic effects are based are no longer valid. Thus longitudinal assays of healthy cells, such as serial assessment of the fluorescence emission of transgenically-expressed proteins, provide more accurate estimates of cell death than do single-time-point anatomical or biochemical assays of the number of dying neurons. More accurate estimates of death rates in vitro will increase the translatability of preclinical studies of neuroprotection and neurotoxicity.
... The fluorescent dye 2′,7′-dichlorodihydro-FDA (DC-FDA) was used to monitor ROS formation. 39,40 Like FDA, DC-FDA is membrane-permeable and becomes deacetylated by intracellular esterases to a membrane-impermeable product. The latter is oxidized by ROS, especially hydrogen peroxide, forming the fluorescent product. ...
The use of metal nanoparticles (NPs) as antimicrobial agents has become a promising alternative to the problem of antibiotic-resistant bacteria and other applications. Silver nanoparticles (AgNPs) are well-known as one of the most universal biocide compounds. However, selenium nanoparticles (SeNPs) recently gained more attention as effective antimicrobial agents. This study aims to investigate the antibacterial activity of SeNPs with different surface coatings (BSA-coated, chitosan-coated, and undefined coating) on the Gram-negative Stenotrophomonas bentonitica and the Gram-positive Lysinibacillus sphaericus in comparison to AgNPs. The tested NPs had similar properties, including shape (spheres), structure (amorphous), and size (50-90 nm), but differed in their surface charge. Chitosan SeNPs exhibited a positive surface charge, while the remaining NPs assayed had a negative surface charge. We have found that cell growth and viability of both bacteria were negatively affected in the presence of the NPs, as indicated by microcalorimetry and flow cytometry. Specifically, undefined coating SeNPs displayed the highest percentage values of dead cells for both bacteria (85-91%). An increase in reactive oxygen species (ROS) production was also detected. Chitosan-coated and undefined SeNPs caused the highest amount of ROS (299.7 and 289% over untreated controls) for S. bentonitica and L. sphaericus, respectively. Based on DNA degradation levels, undefined-SeNPs were found to be the most hazardous, causing nearly 80% DNA degradation. Finally, electron microscopy revealed the ability of the cells to transform the different SeNP types (amorphous) to crystalline SeNPs (trigonal/monoclinical Se), which could have environmentally positive implications for bioremediation purposes and provide a novel green method for the formation of crystalline SeNPs. The results obtained herein demonstrate the promising potential of SeNPs for their use in medicine as antimicrobial agents, and we propose S. bentonitica and L. sphaericus as candidates for new bioremediation strategies and NP synthesis with potential applications in many fields.
... Photographic images were captured using a mobile camera (Asus, Zenfone 2 Laser, Taiwan). The cell viability test was carried out using fluorescein diacetate and propidium iodide dyes to stain live and dead cells (Jones and Senft, 1985). After staining, samples were observed under a fluorescence microscope (Nikon TiS, Japan). ...
Antibiotics, an emerging pollutant in domestic sewage, may likely to impact the performance of biological treatment plants; hence, assessing the health and quality of sludge biomass is essential. This study investigated the impact of 0, 500, 1000, and 2000 mg/L of amoxicillin intravenous form (AMX) on aerobic biomass and assessed its recovery potential when AMX has been stopped in the influent. Reactors have been operated for 43 d with AMX, then another 35 d without AMX in the feed. AMX stressed reactors have exhibited 23-74% COD removal during stressed phase operation, compared to 93% for the control reactor. A noticeable decrease in biomass activity, cell viability, and enzyme activities due to AMX stress have been observed. ARDRA has revealed 52-80% similarity in microbial community structure of control and AMX fed reactors. During the recovery phase, all reactors have exhibited 93% COD removal with increased biomass activity, cell viability, and enzyme activities. ARDRA has displayed 68-94% similarity between control and AMX stressed biomass. This study has demonstrated complete recovery of sludge biomass when AMX has been discontinued in the feed. SVI, ISV, and biomass activity have been identified as the preliminary test. In addition, cell viability, enzyme activity, and ARDRA have been identified as confirmation tests in assessing the health of sludge biomass.
... Cell viability of selected EFs was evaluated using a confocal equipment (Carl Zeiss, Model LSM800, Munich, Germany). Cells were determined by staining with fluorescein diacetate (CFDA) and propidium iodide (PI) (Jones & Senft, 1985). Briefly, 1 cm 2 of fermented EF was collocated with 100 µL of CFDA solution (0.5% w/v in PBS) for 10 min, and then EF was placed with 15 µL of PI (0.002% w/v in PBS) for another 10 min. ...
This study aimed to develop and characterize bioactive edible films (EFs) based on LAB-fermented whey solution and potato starch and evaluate their physical, mechanical, and probiotic count during storage. Fermented whey solutions with Lactobacillus acidophilus and Lactobacillus rhamnosus were mixed with potato starch solution (1:1 v/v) to produce EFs, which were evaluated in their moisture, color, thickness, tensile strength, water vapor permeability, and probiotic load at the beginning and after 14 days of storage (4 ± 1 °C) using PET and LLDPE as secondary packages. Selected EFs were evaluated in their antimicrobial activity against Escherichia coli and their viable cell count and structure using laser scanning confocal (LSC) and environmental scanning electronic micrographs (ESEM), respectively. The thickness (37.5–62.5%) and tensile strength (86.4–136.4%) of EFs increased by adding fermented whey in the film formulation (compared to control EFs), while moisture, color parameters, and elongation at break were not affected. The probiotic count obtained after the gastrointestinal process was higher (1.64–1.82 log) than the obtained in not digested EFs, indicating a liberation of trapped microorganisms during the gastrointestinal process. In general, the package did not affect the mechanical properties of EFs. The probiotic count was not affected by the package and storage time, showing a similar viable count in both plate counting and LSC. Micrographs indicated that LAB-fermented whey EFs showed smooth surfaces without breakups; however, after storage, a reduction in thickness and a fractured surface were observed. Stored LAB-fermented EFs showed antimicrobial activity (21–22 mm inhibition zone) against E. coli. Fermented EFs showed adequate stability to be applied in food products.
... Live/dead staining [18] was conducted to evaluate the viability of the spheroids. Harvested spheroids were washed three times with 0.1 M PBS, then transferred to a Corning ® 24-well plate. ...
Three-dimensional cell culture models are increasingly adopted as preferred pre-clinical drug testing platforms, as they circumvent limitations associated with traditional monolayer cell cultures. However, many of these models are not fully characterized. This study aimed to characterize a BT-20 triple-negative breast carcinoma spheroid model and assess its susceptibility to doxorubicin in comparison to a monolayer model. Spheroids were developed using the liquid overlay method. Phenotypic attributes were analyzed by characterizing changes in size, gross morphology, protein content, metabolic activity, hypoxic status, and cell–cell junctions. The cytotoxic range of doxorubicin in monolayers was determined using the sulforhodamine B assay, and the comparative effect of toxic and sub-toxic concentrations was assessed in both spheroids and monolayers. Similar to the in vivo microenvironment, spheroids had a heterogeneous spatial cytoarchitecture, inherent hypoxia and strong adherens junctions. Doxorubicin induced dose-dependent cytotoxicity in monolayers (IC25: 130 nM, IC50: 320 nM and IC75: 1580 nM); however, these concentrations did not alter the spheroid size or acid phosphatase activity. Only concentrations ≥6 µM had any effect on spheroid integrity. In comparison to monolayers, the BT-20 spheroid model has decreased sensitivity to doxorubicin and could serve as a better model for susceptibility testing in triple-negative breast cancer.
... Giardia trophozoites (1 × 10 4 ) of clones VSP1267, VSP417 and VSPH7 were incubated with 50 nM of its corresponding mAbs (7F5, 7C2 and G10/4, respectively) for 72 h; then, 100 µl were used to analyse viability by propidium iodide/fluorescein diacetate staining 59 . The other 100 µl was used to count the total cell number in a haemocytometer. ...
The genomes of most protozoa encode families of variant surface antigens. In some parasitic microorganisms, it has been demonstrated that mutually exclusive changes in the expression of these antigens allow parasites to evade the host’s immune response. It is widely assumed that antigenic variation in protozoan parasites is accomplished by the spontaneous appearance within the population of cells expressing antigenic variants that escape antibody-mediated cytotoxicity. Here we show, both in vitro and in animal infections, that antibodies to Variant-specific Surface Proteins (VSPs) of the intestinal parasite Giardia lamblia are not cytotoxic, inducing instead VSP clustering into liquid-ordered phase membrane microdomains that trigger a massive release of microvesicles carrying the original VSP and switch in expression to different VSPs by a calcium-dependent mechanism. This novel mechanism of surface antigen clearance throughout its release into microvesicles coupled to the stochastic induction of new phenotypic variants not only changes current paradigms of antigenic switching but also provides a new framework for understanding the course of protozoan infections as a host/parasite adaptive process.
... Cell viability was determined by flow cytometry of cell suspensions obtained by dissociation of retinal explants, in accordance with the technique described in Jones and Senft (1985), using fluorescein diacetate (FDA, Sigma) and propidium iodide (PI, Sigma). Non-fluorescent FDA is a membrane-permeable esterase substrate that is cleaved by the esterase activity of living cells and gives rise to fluorescein, which emits green fluorescence and is retained within viable cells. ...
During development microglia colonize the central nervous system (CNS) and play an important role in programmed cell death, not only because of their ability to remove dead cells by phagocytosis, but also because they can promote the death of neuronal and glial cells. To study this process, we used as experimental systems the developing in situ quail embryo retina and organotypic cultures of quail embryo retina explants (QEREs). In both systems, immature microglia show an upregulation of certain inflammatory markers, e.g., inducible NO synthase (iNOS), and nitric oxide (NO) under basal conditions, which can be further enhanced with LPS-treatment. Hence, we investigated in the present study the role of microglia in promoting ganglion cell death during retinal development in QEREs. Results showed that LPS-stimulation of microglia in QEREs increases (i) the percentage of retinal cells with externalized phosphatidylserine, (ii) the frequency of phagocytic contacts between microglial and caspase-3-positive ganglion cells, (iii) cell death in the ganglion cell layer, and (iv) microglial production of reactive oxygen/nitrogen species, such as NO. Furthermore, iNOS inhibition by L-NMMA decreases cell death of ganglion cells and increases the number of ganglion cells in LPS-treated QEREs. These data demonstrate that LPS-stimulated microglia induce ganglion cell death in cultured QEREs by a NO-dependent mechanism. The fact that phagocytic contacts between microglial and caspase-3-positive ganglion cells increase suggests that this cell death might be mediated by microglial engulfment, although a phagocytosis-independent mechanism cannot be excluded.
... To assess viability of isolated myenteric cells, a two-color live-dead assay using fluorescein diacetate (FDA, F7378, Sigma-Aldrich) and propidium iodide (PI, 537059, Sigma-Aldrich) was performed (Jones and Senft, 1985). In this assay, FDA serves as a viability probe. ...
The enteric nervous system (ENS) is a complex neuronal network organized in ganglionated plexuses that extend along the entire length of the gastrointestinal tract. Largely independent of the central nervous system, the ENS coordinates motility and peristalsis of the digestive tract, regulates secretion and absorption, and is involved in immunological processes. Electrophysiological methods such as the patch-clamp technique are particularly suitable to study the function of neurons as well as the biophysical parameters of the underlying ion channels under both physiological and pathophysiological conditions. However, application of the patch-clamp method to ENS neurons remained difficult because they are embedded in substantial tissue layers that limit access to and targeted manipulation of these cells. Here, we present a robust step-by-step protocol that involves isolation of ENS neurons from adult mice, culturing of the cells, their transfection with plasmid DNA, and subsequent electrophysiological characterization of individual neurons in current-clamp and voltage-clamp recordings. With this protocol, ENS neurons can be prepared, transfected, and electrophysiologically characterized within 72 h. Using isolated ENS neurons, we demonstrate the feasibility of the approach by functional overexpression of recombinant voltage-gated Na V 1.9 mutant channels associated with hereditary sensory and autonomic neuropathy type 7 (HSAN-7), a disorder characterized by congenital analgesia and severe constipation that can require parenteral nutrition. Although our focus is on the electrophysiological evaluation of isolated ENS neurons, the presented methodology is also useful to analyze molecules other than sodium channels or to apply alternative downstream assays including calcium imaging, proteomic and nucleic acid approaches, or immunochemistry.
... In recent years, fluorescence labeling technology has gained much attention due to its outstanding advantages, including rapid response, simple operation, high sensitivity, ease of quantification, etc. [15][16][17][18][19][20][21]. The frequently used fluorescent reagents capable of realizing the discrimination between live and dead cells include SYTOX Green nucleic acid stain [22,23], propidium iodide (PI) [24], rhodamine 123 [25], fluorescein derivatives [26][27][28], calcein acetoxymethyl ester [29], RedDot2 [30], and carbon dots [31][32][33][34][35][36][37][38]. Most of these reagents selectively stain the dead cells via penetrating compromised cell membranes and staining the cell nucleus (e.g., RedDot2). ...
Rapid and accurate differentiation between live and dead cells is highly desirable for the evaluation of cell viability. Here, we report the application of the orange-emitting sulfur-doped organosilica nanodots (S-OSiNDs) for ultrafast (30 s), ultrasensitive (1 μg/mL), and universal staining of the dead bacterial, fungal, and mammalian cells but not the live ones, which satisfies the requirements of a fluorescent probe that can specifically stain the dead cells. We further verify that the fluorescence distribution range of S-OSiNDs (which are distributed in cytoplasm and nucleus) is much larger than that of the commercial dead/fixed cell/tissue staining dye RedDot2 (which is distributed in the nucleus) in terms of dead mammalian cell staining, indicating that S-OSiNDs possess a better staining effect of dead cells than RedDot2. Overall, S-OSiNDs can be used as a robust fluorescent probe for ultrafast and accurate discrimination between dead and live cells at a single cell level, which may find a variety of applications in the biomedical field.
... The key idea of the approach is to use a fluorescent dye that can be detected by both fluorescent microscopy and imaging MS. We chose fluorescein diacetate (FDA), a compound widely used in cell biology to assess cell viability (Mckinney, Spillane and Pearce, 1964;Jones and Senft, 1985), because it has high fluorescence, high mass spectrometry response, has no reported metabolic effects, and is fluorescent only when intracellular. In its esterified form, the compound is cell-permeable and not fluorescent. ...
Imaging mass spectrometry (MS) is becoming increasingly applied for single-cell analyses. Multiple methods for imaging MS-based single-cell metabolomics were proposed, including our recent method SpaceM. An important step in imaging MS-based single-cell metabolomics is the assignment of MS intensities from individual pixels to single cells. In this process, referred to as pixel-cell deconvolution, the MS intensities of regions sampled by the imaging MS laser are assigned to the segmented single cells. The complexity of the contributions from multiple cells and the background, as well as lack of full understanding of how input from molecularly-heterogeneous areas translates into mass spectrometry intensities make the cell-pixel deconvolution a challenging problem.
Here, we propose a novel approach to evaluate pixel-cell deconvolution methods by using a molecule detectable both by mass spectrometry and fluorescent microscopy, namely fluorescein diacetate (FDA). FDA is a cell-permeable small molecule that becomes fluorescent after internalisation in the cell and subsequent cleavage of the acetate groups. Intracellular fluorescein can be easily imaged using fluorescence microscopy. Additionally, it is detectable by matrix-assisted laser desorption/ionisation (MALDI) imaging MS. The key idea of our approach is to use the fluorescent levels of fluorescein as the ground truth to evaluate the impact of using various pixel-cell deconvolution methods onto single-cell fluorescein intensities obtained by the SpaceM method.
Following this approach, we evaluated multiple pixel-cell deconvolution methods, the 'weighted average' method originally proposed in the SpaceM method as well as the novel 'linear inverse modelling' method. Despite the potential of the latter method in resolving contributions from individual cells, this method was outperformed by the weighted average approach. Using the ground truth approach, we demonstrate the extent of the ion suppression effect which considerably worsens the pixel-cell deconvolution quality. For compensating the ion suppression, we propose a novel data-driven approach. We show that compensating the ion suppression effect in a single-cell metabolomics dataset of co-cultured HeLa and NIH3T3 cells considerably improved the separation between both cell types. Finally, using the same ground truth, we evaluate the impact of drop-outs in the measurements and discuss the optimal filtering parameters of SpaceM processing steps before pixel-cell deconvolution.
... Calcein AM is a dye that permeates live cells and is converted to fluorescent calcein by intracellular esterases and therefore acts as a live cell dye for viability measurements [247]. PI penetrates dead and dying cell membranes, once inside cells PI intercalates with DNA to become fluorescent; however, it cannot penetrate live cells and can therefore be used a cell viability marker [248,249]. ...
Parkinson’s disease is associated with the aberrant aggregation of α-synuclein within neurons. Although the causes of this process are still unclear, post-translational modifications of α-synuclein are likely to play a modulatory role. Since α-synuclein is constitutively N-terminally acetylated, we investigated how this post-translational modification alters the aggregation behaviour of this protein. By applying a three-pronged aggregation kinetics approach, we observed that N-terminal acetylation results in a reduced rate of lipid-induced aggregation and in a slowing down of both elongation and fibril-catalysed aggregate proliferation. An analysis of the amyloid fibrils produced by the aggregation process revealed different morphologies for the acetylated and non-acetylated forms in both the lipid-induced aggregation and seed-induced aggregation assays. In addition, we found that fibrils formed by acetylated α-synuclein possess a lower β-sheet content. These findings indicate that N-terminal acetylation of α-synuclein alters its lipid-dependent aggregation behaviour, reduces its rate of in vitro aggregation, and affects the structural properties of its fibrillar aggregates. We then investigated how this modification affects the α-synuclein mutants associated with familial Parkinson’s disease. We found that all N-terminal acetylated mutants were capable of forming seeding-competent, amyloid-like aggregates in the presence of lipid vesicles. These results are relevant as lipid membranes could stimulate the initial nucleation process that leads to the aggregation of α-synuclein in vivo. In perspective, the set of assays that we have developed can be taken forward and used to investigate how other post-translational modifications can impact the behaviour of α-synuclein.
... Double staining of propidium iodide (PI) and fluorescein diacetate (FDA) was performed to detect neuronal death/ viability (Jones and Senft, 1985;Zheng et al., 2012). Briefly, cultures were rinsed with an extracellular solution and incubated with FDA (5.0 μM) and PI (2.0 μM) for 30 min. ...
Background: We and others have previously demonstrated that glycine is neuroprotective in cerebral ischemia-reperfusion injury. But glycine has low permeability to the blood–brain barrier (BBB). To deliver glycine into the ischemic brain to confer neuroprotection, we designed a novel glycine-containing and BBB-permeable tripeptide, the H-glycine-cysteine-phenylalanine-OH (GCF).
Methods: For the synthesis of GCF, phenylalanine was included to increase the BBB permeability of the tripeptide. Cysteine was conjugated with glycine to enable the release of glycine from GCF. With the use of immunofluorescence labeling and HPLC assays, we measured the distribution and level of GCF. We used TTC labeling, LDH release, and MTT assays to evaluate the neuroprotective effect of GCF.
Results: Following intravenous injection in a rat model of cerebral ischemia-reperfusion injury, GCF was intensively distributed in the ischemic neurons. Intravenous injection of GCF, but not the non-cleavable acetyl-GCF, resulted in the elevation of glycine in the ischemic brain. GCF but not acetyl-GC conferred neuroprotection in ischemic stroke animals.
Conclusion: GCF protects against cerebral ischemia-reperfusion injury in the rat. In contrast to peptide drugs that exert therapeutic effect by interfering with signaling interaction, GCF acts as a BBB shuttle and prodrug to deliver glycine to confer neuroprotection, representing a novel therapeutic strategy for acute ischemic stroke.
... The produced fluorescein accumulates inside the cell, resulting in green fluorescence when excited. Consequently, compromised cells with a reduced amount of esterase activity exhibit a reduced fluorescence signal compared to healthy cells (Jones and Senft 1985). Although, complementary staining properties are given in case of AO (stains all cells) and FDA (only living cells) it is not feasible to use the two dyes simultaneously in a single assay, since both dyes have similar emissions maxima (526 vs. 520 nm) and consequently, due to spectral overlap their signals cannot be discriminated. ...
Determination of the viability, ratio of dead and live cell populations, of Sulfolobus acidocaldarius is still being done by tedious and material-intensive plating assays that can only provide time-lagged results. Although S. acidocaldarius, an extremophilic Archaeon thriving at 75 °C and pH 3.0, and related species harbor great potential for the exploitation as production hosts and biocatalysts in biotechnological applications, no industrial processes have been established yet. One hindrance is that during development and scaling of industrial bioprocesses timely monitoring of the impact of process parameters on the cultivated organism is crucial—a task that cannot be fulfilled by traditional plating assays. As alternative, flow cytometry (FCM) promises a fast and reliable method for viability assessment via the use of fluorescent dyes. In this study, commercially available fluorescent dyes applicable in S. acidocaldarius were identified. The dyes, fluorescein diacetate and concanavalin A conjugated with rhodamine, were discovered to be suitable for viability determination via FCM. For showing the applicability of the developed at-line tool for bioprocess monitoring, a chemostat cultivation on a defined growth medium at 75 °C, pH 3.0 was conducted. Over the timeframe of 800 h, this developed FCM method was compared to the plating assay by monitoring the change in viability upon controlled pH shifts. Both methods detected an impact on the viability at pH values of 2.0 and 1.5 when compared to pH 3.0. A logarithmic relationship between the viability observed via plating assay and via FCM was observed.
... 59 PI penetrates dead and dying cell membranes, and once inside cells, PI intercalates with DNA to become fluorescent; however, it cannot penetrate live cells and can therefore be used as a cell viability marker. 60,61 Our results using non-differentiated SH-SY5Y cells indicate no significant difference in the cytotoxicity of the PFFs derived from either non-acetylated or acetylated α-synuclein, as assessed by Calcein AM and PI fluorescence (Figure 8). We also tested the effects of stabilized oligomers from both acetylated and non-acetylated α-synuclein on the viability of SH-SY5Y cells ( Figure S4C). ...
Parkinson's disease is associated with the aberrant aggregation of α-synuclein. Although the causes of this process are still unclear, post-translational modifications of α-synuclein are likely to play a modulatory role. Since α-synuclein is constitutively N-terminally acetylated, we investigated how this post-translational modification alters the aggregation behavior of this protein. By applying a three-pronged aggregation kinetics approach, we observed that N-terminal acetylation results in a reduced rate of lipid-induced aggregation and slows down both elongation and fibril-catalyzed aggregate proliferation. An analysis of the amyloid fibrils produced by the aggregation process revealed different morphologies for the acetylated and non-acetylated forms in both lipid-induced aggregation and seed-induced aggregation assays. In addition, we found that fibrils formed by acetylated α-synuclein exhibit a lower β-sheet content. These findings indicate that N-terminal acetylation of α-synuclein alters its lipid-dependent aggregation behavior, reduces its rate of in vitro aggregation, and affects the structural properties of its fibrillar aggregates.
... As previously demonstrated, this method provides reliable, repeatable injuries in the absence of cell death 25 . However, for this investigation, a supplemental viability study was performed using fluorescein diacetate (FDA) and propidium iodide (PI), following standard procedures 70,71 . Briefly, cells were thrice rinsed with isotonic phosphate buffered saline (PBS, pH = 7.4, Gibco), exposed to 25 μM and 7.5 μM of FDA and PI respectively for 5 min, and then imaged in PBS. ...
While clinical observations have confirmed a link between the development of neurodegenerative diseases and traumatic brain injuries (TBI), there are currently no treatments available and the underlying mechanisms remain elusive. In response, we have developed an in vitro pendulum trauma model capable of imparting rapid acceleration injuries to neuronal networks grown on microelectrode arrays within a clinically relevant range of g forces, with real-time electrophysiological and morphological monitoring. By coupling a primary physical insult with the quantification of post-impact levels of known biochemical pathological markers, we demonstrate the capability of our system to delineate and investigate the primary and secondary injury mechanisms leading to post-impact neurodegeneration. Specifically, impact experiments reveal significant, force-dependent increases in the pro-inflammatory, oxidative stress marker acrolein at 24 h post-impact. The elevation of acrolein was augmented by escalating g force exposures (30–200 g), increasing the number of rapidly repeated impacts (4–6 s interval, 3, 5 and 10×), and by exposing impacted cells to 40 mM ethanol, a known comorbidity of TBI. The elevated levels of acrolein following multiple impacts could be reduced by increasing time-intervals between repeated hits. In addition, we show that conditioned media from maximally-impacted cultures can cause cellular acrolein elevation when introduced to non-impact, control networks, further solidifying acrolein’s role as a diffusive-factor in post-TBI secondary injuries. Finally, morphological data reveals post-impact acrolein generation to be primarily confined to soma, with some emergence in cellular processes. In conclusion, this novel technology provides accurate, physical insults with a unique level of structural and temporal resolution, facilitating the investigation of post-TBI neurodegeneration.
Cervical cancer is the fourth most common cancer in women worldwide. Most cases of cervical cancer are caused by the human papillomavirus. Triterpenes, which are secondary metabolites of plants, are known for their antitumor activity. Cordialin A, isolated from the ethanolic extract of Varronia curassavica Jacq. leaves, shows potential for cervical cancer therapy. Cordialin A is a low-polarity molecule, and its incorporation into nanoemulsion systems (NEs) can enhance its cytotoxic effects on tumor cells. The NE, composed of cholesterol, phosphate buffer, the surfactant polyoxyethylene 20-cetyl ether, and soy phosphatidylcholine, was used to encapsulate cordialin A. The droplets of the cordialin A-loaded NE measured 107 nm in size, with a particle distribution index of 0.214, and a ζ-potential of − 20 mV. Cytotoxicity assays showed that the concentration required to inhibit 50% of cell viability was more potent in cordialin A-loaded NE compared to free cordialin A against cervical tumor cell lines. Marked morphological changes indicative of apoptosis and necrosis were observed in cells after 12 h of exposure to both cordialin A and cordialin A-loaded NE. These results suggest that cordialin A-loaded NE has potential for use in the treatment of cervical cancer.
Cordialin A isolated from Varronia curassavica Jacq. loaded in nanoemulsion as potential cytotoxic agent on human cervical tumor cells
Modified synthetic peptides have emerged as an exciting avenue for enhancing therapeutic efficacy and expanding the scope of applications in various disease contexts. Indeed, the inherent tunability of synthetic peptides has facilitated the creation of highly selective and responsive sensors capable of detecting specific analytes with precision. More recently, their unique structural diversity and chemical versatility has been elegantly adapted for use in supramolecular sensing platforms. This Perspective article highlights the synergistic interplay between modified synthetic peptides, therapeutic applications, and the sensing technologies that underscore the interdisciplinary nature of contemporary chemistry.
Araucaria angustifolia is a critically endangered species and its distribution can be affected by an increase in temperature. In this study, we evaluated the effects of heat stress (30°C) on Araucaria angustifolia cell lines responsive (SE1) and non-responsive (SE6) to the development of somatic embryos. The viability of both cell lines was reduced by heat stress and mitochondria were the organelles most affected. Heat stress for 24 h increased the reactive oxygen species (ROS) levels in SE1 cells, followed by a reduction at 48 and 72 h. In SE6 cells, an increase occurred after 24 and 48 h of stress, returning to control levels at 72 h. H2O2 levels were increased after 24 h for both SE1 and SE6 cells, being higher for SE6. Interestingly, at 48 and 72 h, H2O2 levels decreased in SE1 cells, while in SE6, the values returned to the control levels. The respiration of SE6 cells in the presence of oxidisable substrates was inhibited by heat stress, in agreement with the high lipid peroxidation levels. The AaSERK1 gene was identified in both cultures, with greater expression in the SE1 line. Heat stress for 24 and 48 h increased gene expression only in this cell line. The activity of peroxidase, superoxide dismutase and enzymes of the glutathione/ascorbate cycle was increased in both cell lines subjected to heat stress. Catalase activity was increased only in SE6 cells at 72 h of exposure. These results show that responsive SE1 cells can modulate ROS levels more efficiently than SE6 when these cells are stressed by heat. This ability may be related to the maturation capacity of these cells.
Planarians show outstanding regenerative ability due to the proliferation of neoblasts. Hence the method to isolate planarian neoblasts is important to understand the regeneration process. In our previous study, we reported a method to isolate planarian neoblasts of Dugesia japonica using fluorescence‐activated cell sorting (FACS). However, we have not yet succeeded in cultivating these cells even under in vivo conditions after transplantation into X‐ray‐irradiated planarians. This suggests that dissociated cells might enter apoptotic or necrotic states in the process of fluorescent dye staining and sorting. Here, we developed a new method to isolate viable neoblasts which can proliferate in the X‐ray‐irradiated planarians. First, the toxicity of various fluorescence dyes was investigated. All nuclear fluorescent dyes, such as Hoechst 33342, DRAQ5, and DyeCycle, showed more or less toxicity to mammalian culture cells. In contrast, cytoplasmic fluorescent dye for live cells, calcein AM, was less toxic on these cells. Next, we stained the dissociated planarian cells with only calcein AM, and then collected the X‐ray‐sensitive fraction. Although the purity of neoblasts was slightly lower than that of the original staining method (ca. 97% → ca. 89%), the sorted cells could actively proliferate when they were injected into X‐ray‐irradiated planarians. This simple staining and sorting method will provide new opportunities to isolate viable neoblasts and understand regenerating processes.
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Major advances have been made in our understanding of anther developmental processes in flowering plants through a combination of genetic studies, cell biological technologies, biochemical analyses, microarray and high-throughput sequencing-based approaches. In this chapter, we summarize widely used protocols for pollen viability staining, investigation of anther morphogenesis by scanning electron microscopy (SEM), light microscopy of semi-thin sections, ultrathin section-based transmission electron microscopy (TEM), TUNEL (terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate (dUTP) nick end labeling) assay for tapetum programmed cell death, and laser microdissection procedures to obtain specific cells or cell layers for transcriptome analysis.
Background
Neurofibrillary tangles (NFTs) are one of the most common pathological characteristics of Alzheimer's disease (AD). The NFTs are mainly composed of hyperphosphorylated microtubule-associated tau. Thus, recombinant tau is urgently required for the study of its fibrillogenesis and its associated cytotoxicity.
Methods and Results
Heterologous expression, purification, and fibrillation of the microtubule-binding domain (MBD) of tau (tau&MBD) were performed. The tau&MBD was heterologously expressed in E. coli. Ni-chelating affinity (Ni-NTA) chromatography was then performed to purify the target protein. Thereafter, tau&MBD was systematically identified using the SDS-PAGE, western blot and MALDI-TOF MS methods. The aggregation propensity of the tau&MBD was explored by both the thioflavin T fluorescence and atomic force microscopy experiments.
Conclusions
The final yield of the recombinant tau&MBD was ~20 mg·L⁻¹. It is shown that Tau&MBD self-assembled into the typical fibrils. Finally, the in vitro cytotoxicity of tau&MBD aggregates was validated using PC12 cells. The heterologously expressed tau in this study can be further used in the investigation of the biophysical and cellular cytotoxic properties of tau.
Trauma-induced Alzheimer’s disease (AD) is rapidly emerging as a major consequence of Traumatic Brain Injuries (TBI), with devastating social and economic impacts. Unfortunately, few treatment options are currently available due...
Protoplasts, which are plant cells with their cell walls removed, have been used for decades in plant research and have been instrumental in genetic transformation and the study of various aspects of plant physiology and genetics. With the advent of synthetic biology, these individualized plant cells are fundamental to accelerate the "design-build-test-learn" cycle, which is relatively slow in plant research. Despite their potential, challenges remain to expanding the use of protoplasts in synthetic biology. The capacity of individual protoplasts to hybridize to form new varieties, and to regenerate from single cells, creating individuals with new features is underexplored. The main objective of this review is to discuss the use of protoplasts in plant synthetic biology and to highlight the challenges to exploiting protoplast technologies in this new "age of synthetic biology".
Fibrin-based hydrogels are used as scaffolds in tissue engineering and regenerative medicine due to their biocompatibility, low cell toxicity, autologous production, and relevance for wound healing and clot formation. The availability of fibrinogen as well as its unique mechanical behavior exhibiting nonlinear elasticity makes it suitable for the fabrication of hydrogels. However, the broad application of fibrin hydrogels in biomaterials still faces challenges in terms of gel shrinkage and degradation processes. This can be addressed through the modulation of the hydrogels'r chemical and mechanical properties. In the present work, it is demonstrated that fibrin-based hydrogels with adjustable mechanical properties and controllable degradation profiles can be fabricated through the addition of fibrin-binding peptides. The cyclic peptide X2CXYYGTCLX (Tn7) is used, binding to fibrin by noncovalent supramolecular interactions. These new hydrogels exhibit no toxicity and reduced degradation rate at the same time supporting cell proliferation. Tn7 peptides significantly increase the Young's Modulus and mechanical stiffness as well as fibrin fiber thickness and inter-fiber crosslinking in hydrogels. In conclusion, hydrogels with optimized mechanical properties and controllable degradation profiles that can be advantageous for further approaches in tissue regeneration, cell-based therapies, or clinical treatment options are produced.
Thymol (2-isopropyl-5-methylphenol; Thy) is a monoterpene phenolic phytocompound with medicinal properties; however, its impact on osteogenesis is yet to be thoroughly investigated. Its distribution is often hampered because of its intricate hydrophobic structure, which reduces its bioavailability. In this study, we synthesized a drug delivery vehicle using semi-interpenetrating polymer network (SIPN) hydrogels containing sodium alginate and poly(2-ethyl-2-oxazoline) (SA/Pox) loaded with Thy at varying concentrations (100, 150, and 200 μM). Subsequently, they were coated with chitosan (CS) to increase bioactivity and for sustained and prolonged release of Thy. Thy-loaded CS-coated SIPN hydrogels (SA/Pox/CS-Thy) were developed using ionic gelation and polyelectrolyte-complexation techniques. The addition of CS to hydrogels enhanced their physicochemical and material properties. These hydrogels were cytofriendly toward mouse mesenchymal stem cells (mMSCs). When mMSCs were cultured on hydrogels, Thy stimulated osteoblastic differentiation, as evidenced by calcium deposits at the cellular level. The expression of RUNX2, a key bone transcriptional factor, and other differentiation biomarkers was significantly enhanced in mMSCs cultured on SA/Pox/CS-Thy hydrogels. Notably, Thy in the SA/Pox/CS hydrogels significantly activated the TGF-β/BMP signaling pathway, which is involved in osteogenesis. A rat tibial bone defect model system revealed that the incorporation of Thy into SA/Pox/CS hydrogels augmented bone regeneration. Thus, sustained and prolonged release of Thy from the SA/Pox/CS hydrogels promoted osteoblast differentiation in vitro and bone formation in vivo. These findings shed light on the effect of Thy bioavailability in fostering osteoblast differentiation and its prospective application in bone rejuvenation.
Although the handling and exploitation of cyanobacteria is associated with some challenges, these phototrophic bacteria offer great opportunities for innovative biotechnological processes. This chapter covers versatile aspects of working with cyanobacteria, starting with up-to-date in silico and in vitro screening methods for bioactive substances. Subsequently, common conservation techniques and vitality/viability estimation methods are compared and supplemented by own data regarding the non-invasive vitality evaluation via pulse amplitude modulated fluorometry. Moreover, novel findings about the influence the state of the pre-cultures have on main cultures are presented. The following sub-chapters deal with different photobioreactor-designs, with special regard to biofilm photobioreactors, as well as with heterotrophic and mixotrophic cultivation modes. The latter topic provides information from literature on successfully enhanced cyanobacterial production processes, augmented by own data.Graphical AbstractKeywordsCell characterizationCyanobacteriaMixo-/Heterotrophic cultivationPhotobioreactorsStrain conservation
Imaging mass spectrometry (MS) is becoming increasingly applied for single-cell analyses. Multiple methods for imaging MS-based single-cell metabolomics were proposed, including our recent method SpaceM. An important step in imaging MS-based single-cell metabolomics is the assignment of MS intensities from individual pixels to single cells. In this process, referred to as pixel-cell deconvolution, the MS intensities of regions sampled by the imaging MS laser are assigned to the segmented single cells. The complexity of the contributions from multiple cells and the background, as well as lack of full understanding of how input from molecularly-heterogeneous areas translates into mass spectrometry intensities make the cell-pixel deconvolution a challenging problem. Here, we propose a novel approach to evaluate pixel-cell deconvolution methods by using a molecule detectable both by mass spectrometry and fluorescent microscopy, namely fluorescein diacetate (FDA). FDA is a cell-permeable small molecule that becomes fluorescent after internalisation in the cell and subsequent cleavage of the acetate groups. Intracellular fluorescein can be easily imaged using fluorescence microscopy. Additionally, it is detectable by matrix-assisted laser desorption/ionisation (MALDI) imaging MS. The key idea of our approach is to use the fluorescent levels of fluorescein as the ground truth to evaluate the impact of using various pixel-cell deconvolution methods onto single-cell fluorescein intensities obtained by the SpaceM method. Following this approach, we evaluated multiple pixel-cell deconvolution methods, the ‘weighted average’ method originally proposed in the SpaceM method as well as the novel ‘linear inverse modelling’ method. Despite the potential of the latter method in resolving contributions from individual cells, this method was outperformed by the weighted average approach. Using the ground truth approach, we demonstrate the extent of the ion suppression effect which considerably worsens the pixel-cell deconvolution quality. For compensating the ion suppression individually for each analyte, we propose a novel data-driven approach. We show that compensating the ion suppression effect in a single-cell metabolomics dataset of co-cultured HeLa and NIH3T3 cells considerably improved the separation between both cell types. Finally, using the same ground truth, we evaluate the impact of drop-outs in the measurements and discuss the optimal filtering parameters of SpaceM processing steps before pixel-cell deconvolution.
L'utilisation de nanomatériaux est de plus en plus fréquente dans l'industrie pour améliorer la qualité des produits grâce à leurs propriétés particulières. Par exemple, les silices amorphes de synthèse sont utilisées dans les cosmétiques, l'alimentation humaine et l'agroalimentaire, ainsi que dans les pneumatiques pour leurs propriétés abrasives, de renforcement ou d'épaississement. Ces utilisations très diverses exposent les travailleurs et la population à des risques potentiels à plus ou moins long terme, qui doivent être évalués, dans un contexte plus global de multiexpositions, tel que rencontré dans les populations humaines. La multiplicité des scenarii à explorer rend l'utisation des modèles animaux problématique, pour des raisons éthiques et économiques. C'est pourquoi nous utiliserons des systèmes in vitro de macrophages (lignées RAW264.7 et J774A.1, macrophages primaires murins) ce type cellulaire étant une cible clée en toxicologie des matériaux particulaires. Les effets de différents scénarii d'exposition seront étudiés : l'exposition aigüe pendant 24h, l'exposition chronique pendant plusieurs jours consécutifs, puis la récupération qui correspond à l'un ou l'autre des scénarii suivis de quelques jours sans nanomatériaux, qui est un paramètre fondamental en nanotoxicologie. Les expériences réalisées seront basées sur des tests de viabilité et des tests fonctionnels (phagocytose, dosages de NO et d'espèces oxydantes, de production de cytokines pro et anti-inflammatoires) grâce à la cytométrie en flux, la spectrophotométrie, et la microscopie.
p>Previous research has demonstrated the effectiveness of applying a simple ageing treatment over the ASTM standard passivation treatment to reduce metal ion release from Ti-6Al-4V alloy into bovine serum solution. The present study aims to further understand the mechanisms behind the improved dissolution resistance exhibited by the aged Ti-6Al-4V alloy using a number of sophisticated surface analysis techniques, X-ray photoelectron spectroscopy and atomic force microscopy. Furthermore, osteoblastic cell culture and functional genomic studies have been employed to fully characterise the biological performance of the treated implants.
This study has demonstrated that the passivation treatment results in the elimination of vanadium from the oxide layers. The O/Ti ratio of the passivated sample significantly changes with immersion time in serum, indicating that further oxidation and dissolution processes were occurring. In contrast, the aged oxide surface is more stable and possesses hydroxylated groups, which are thought to play an important role in the reaction with serum proteins. In addition, this research has observed that the side effects of Al<sub>2</sub>O<sub>3</sub> grit-blasting and heat treatment associated with the hydroxyapatite plasma-spraying coating cause the change in the Al% surface composition and the loss of chemisorbed H<sub>2</sub>O groups; this in turn, affects the metal ion dissolution behaviour of the treated implant. Furthermore, a potentiodynamic test was used to assess the nature of the passive film; the aged surface showed better dissolution resistance and was less affected by bovine serum solution than other treatments.
Based on the findings of this study the aged surface has demonstrated much improved dissolution resistance, which is reflected in its biological performance. Moreover, the genocompatibility test is introduced in this study to complement existing biocompatability tests and to provide further detailed information on cell-implant interface reactions.</p
Electroporation is a fundamental technique for applications in biotechnology. To date, the ongoing research on cell membrane electroporation has explored its mechanism, principles and potential applications. Therefore, in this review, we first discuss the primary electroporation mechanism to help establish a clear framework. Within the context of its principles, several critical terms are highlighted to present a better understanding of the theory of aqueous pores. Different degrees of electroporation can be used in different applications. Thus, we discuss the electric factors (shock strength, shock duration, and shock frequency) responsible for the degree of electroporation. In addition, finding an effective electroporation detection method is of great significance to optimize electroporation experiments. Accordingly, we summarize several primary electroporation detection methods in the following sections. Finally, given the development of micro- and nano-technology has greatly promoted the innovation of microfluidic-based electroporation devices, we also present the recent advances in microfluidic-based electroporation devices. Also, the challenges and outlook of the electroporation technique for cell membrane electroporation are presented.
The influence of four factors on the dye exclusion test for cell viability was investigated. Eosin concentration, serum concentration, cell concentration and staining time were each varied in turn to assess their effects on the number of rabbit leucocytes staining in damaged and undamaged suspensionsStained cell counts tended to be high when high concentrations of stain and/or low concentrations of serum were used, and vice versa, irrespective of whether suspensions were initially damaged or not. The stained cell counts of undamaged suspensions were not affected by staining times up to 90 min. In damaged suspensions the proportion of stained cells increased with the staining time. Stained cell counts tended to be high when very low concentrations of cells were used.