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Analysis of CFSE signal decay upon cell proliferation. a CFSE passively diffuses into the cells and covalently binds to free amine residues. As cells divide, each daughter cell holds half of the CFSE content present in the mother cell. b HEK 293 cells were stained with CFSE and cultured for up to 7 days. Cells were harvested and fixed at different time points and the CFSE fluorescence signal was measured. Consecutive cell divisions lead to progressive CFSE signal decay in the cell population. The experiment was carried out with three technical replicates. c CFSE MFI as function of time reveals the exponential CFSE signal decay. d The inverse of CFSE MFI values (MFI −1 ) plotted as function of time renders the curve into a sigmoid, similar to the conventional growth curves
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Objective:
Cell growth curves constitute one of the primary assays employed to analyze cell proliferation dynamics of in vitro cultured cells under specific culture conditions. From the cell growth curve, it is possible to assess the behavior of proliferating cells under different conditions, such as drug treatment and genomic editions. Traditiona...
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... the presence or absence of 20 µg/mL Mitomycin C, was carried out using the t test. Pereira et al. BMC Res Notes (2020) 13:57 Results HEK 293 cells were stained with CFSE, seeded onto several wells and cultured for 7 days. Cells were harvested at different time points and the CFSE MFI was determined using the Accuri C6 Cytometer (BD Biosciences; Fig. 1b). The CFSE MFI from each time point was plotted as a function of time rendering an exponential descendent curve (Fig. 1c). Then, the inverse of CFSE MFI measurements were plotted as a function of time, transforming the descendent exponential plot into an ascendant plot and fitting very closely the conventional cell growth curve (Fig. ...
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... 13:57 Results HEK 293 cells were stained with CFSE, seeded onto several wells and cultured for 7 days. Cells were harvested at different time points and the CFSE MFI was determined using the Accuri C6 Cytometer (BD Biosciences; Fig. 1b). The CFSE MFI from each time point was plotted as a function of time rendering an exponential descendent curve (Fig. 1c). Then, the inverse of CFSE MFI measurements were plotted as a function of time, transforming the descendent exponential plot into an ascendant plot and fitting very closely the conventional cell growth curve (Fig. ...
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... Fig. 1b). The CFSE MFI from each time point was plotted as a function of time rendering an exponential descendent curve (Fig. 1c). Then, the inverse of CFSE MFI measurements were plotted as a function of time, transforming the descendent exponential plot into an ascendant plot and fitting very closely the conventional cell growth curve (Fig. ...
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... address this question, the MFI of these last two time points were compared using Mitomycin C to halt cell proliferation. The number of cells did not increase upon Mitomycin C treatment and no CFSE signal decay was observed, confirming that CFSE decay matches to cell proliferation even after 7 days under cell culture conditions (Additional file 1: Figure S1). Since CFSE signal decays over time, this method may have limitations for longer-term studies, and this validation is requested for these cases. ...
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... by cell loss during cells harvesting, which directly affects the total number of counted cells. Moreover, cellular debris and cell clumps increase the underestimation of total number of cells in cell-counting based growth curves. On the other hand, fluorescence-based method constitutes the usage of fluorescence cell tracers to stain cells (Fig. 1) and tracking proliferating cells by analysis of fluorescence decay [13]. As a result, the fluorescence of a cell population decreases as a function of time as cells proliferate, allowing this methodology to be employed to assess cell proliferation and to determine cell lines doubling time [11,14]. This method is based on assessing the ...
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Citations
... Single cells were identified by plotting FSC-A vs. FSC-H, and the CFSE median fluorescence intensity (MFI) of at least 8000 single cells was determined. Exponential growth curves were generated by plotting 1/MFI as a function of time [31]. The function N(t) = N(0) × e kt was fitted to the data by non-linear regression (SigmaPlot 12.5, Systat Software Inc., San Jose, CA, USA) to determine the growth rate k and the doubling time, DT (DT = ln2/k). ...
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... Fluorescence assay has been implemented to monitor the growth of cultured cells under specific culture conditions (Fuge et al. 2017;Orekhova et al. 2018;Pereira et al. 2020;Zhou et al. 2018;Quah and Parish 2012;Begum et al. 2013). For example, fluorescence-based method using the carboxyfuorescein diacetate succinimidyl ester (CFSE) fuorescence decay over time was deployed to record the cell growth of adherent cells to replace the counting-based method, exhibiting less biased, lower variation, better reliability, and higher accuracy than counting-based methods (Pereira 2020). ...
... Fluorescence assay has been implemented to monitor the growth of cultured cells under specific culture conditions (Fuge et al. 2017;Orekhova et al. 2018;Pereira et al. 2020;Zhou et al. 2018;Quah and Parish 2012;Begum et al. 2013). For example, fluorescence-based method using the carboxyfuorescein diacetate succinimidyl ester (CFSE) fuorescence decay over time was deployed to record the cell growth of adherent cells to replace the counting-based method, exhibiting less biased, lower variation, better reliability, and higher accuracy than counting-based methods (Pereira 2020). Also, the use of CFSE to measure lymphocyte proliferation by flow cytometry is a well-established approach for evaluating lymphocyte responses Parish 2012, Quah et al. 2007). ...
The measurement of fungal cell growth in submerged culture systems containing insoluble compounds is essential yet difficult due to the interferences from the insoluble compounds like biopolymers. Here, we developed a fluorescent strategy based on chitosan-modified fluorescein isothiocyanate (GC-FITC) to monitor the cell growth of lignocellulosic fungi cultivated on biopolymers. GC-FITC could stain only lignocellulosic fungi (T. reesei, P. oxalicum, A. nidulans, and N. crassa), but not biopolymers (cellulose, xylan, pectin, or lignin), excluding the interferences from these insoluble biopolymer. Moreover, a linear relationship was observed between the fluorescence intensity of GC-FITC absorbed by lignocellulosic fungi and the biomass of lignocellulosic fungi. Therefore, GC-FITC was leveraged to monitor the cell growth of lignocellulosic fungi when using biopolymers like cellulose as the carbon sources, which is faster, more convenient, time-saving and cost-effective than the existing methods using protein/DNA content measurement. GC-FITC offers a powerful tool to detect fungal growth in culture systems with insoluble materials.
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... Cell proliferation assay. The procedure carried out according to the published protocol 48 ...
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... To evaluate the impact of PPO deletion on cell proliferation, the growth rate of PPO-KO and control cell lines in RPMI1640 medium supplemented with 10% FBS was assessed using the carboxyfluorescein succinimidyl ester (CFSE) labeling protocol, where the cell doubling rate negatively correlates with the intensity of intracellular fluorescence of the fluorophore 48 . Surprisingly, the growth rate of PPO-KO clones was similar to that of control cell lines suggesting that the heme synthesis pathway is not required for the cell growth under "traditional" cultivation conditions in vitro, where all nutrients are available in excess (Fig. 2d). ...
In humans, disruptions in the heme biosynthetic pathway are associated with various types of porphyrias, including variegate porphyria that results from the decreased activity of protoporphyrinogen oxidase IX (PPO; E.C.1.3.3.4), the enzyme catalyzing the penultimate step of the heme biosynthesis. Here we report the generation and characterization of human cell lines, in which PPO was inactivated using the CRISPR/Cas9 system. The PPO knock-out (PPO-KO) cell lines are viable with the normal proliferation rate and show massive accumulation of protoporphyrinogen IX, the PPO substrate. Observed low heme levels trigger a decrease in the amount of functional heme containing respiratory complexes III and IV and overall reduced oxygen consumption rates. Untargeted proteomics further revealed dysregulation of 22 cellular proteins, including strong upregulation of 5-aminolevulinic acid synthase, the major regulatory protein of the heme biosynthesis, as well as additional ten targets with unknown association to heme metabolism. Importantly, knock-in of PPO into PPO-KO cells rescued their wild-type phenotype, confirming the specificity of our model. Overall, our model system reveals physiological consequences of the PPO ablation at the cellular level and can serve as a tool to study various aspects of dysregulated heme metabolism associated with variegate porphyria.
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Graphic abstract
... Cell proliferation was evaluated using the carboxyfluorescein diacetate N-succinimidyl ester (CFSE) dilution concept. CFSE is a cellpermeant dye and is equally partitioned to daughter cells during cell division, allowing the indirect assessment of proliferation (Pereira et al., 2020). ...
Ethnopharmacological relevance
The polysaccharides of the millenary mushroom Ganoderma lucidum (GL) have been shown for decades to present anti-tumor activities, but few studies evaluated its importance on cancer stem cells and EMT process. Cancer stem cells (CSC) drive the development of carcinoma and are also involved in cancer treatment failure, being a good target for treatment success. Also, the process of epithelial-mesenchymal transition (EMT) is involved in metastasis and cancer relapse. Besides that, the increasing incidence worldwide of oral squamous cell carcinoma (OSCC) became a public health issue with a high rate of metastasis and poor quality of life for patients during and after treatment.
Aim of the study
to evaluate G. lucidum polysaccharides (GLPS) in vitro effects on OSCC, focusing on hallmarks associated with tumorigenesis using the SCC-9, a squamous cells carcinoma lineage from the tongue.
Materials and methods
SCC-9 cells were treated in vitro for 72h with different GLPS concentrations. The controls cells were maintained with culture media only and cisplatin was used as treatment control. After the treatment period, the cells were evaluated.
Results
GLPS treatment changed cell morphology and granularity, delayed migration, decreased colony, and impaired sphere formation, thereby leading to a non-invasive and less proliferative behavior of tumoral cells. Additionally, GLPS downregulated CSC, EMT, and drug sensitivity (ABC) markers.
Conclusions
These results show that the natural product GLPS has the potential to be an important ally for tongue squamous cell carcinoma treatment, bringing the millenary compound to modern therapy, providing a basis for future studies and the improvement of life quality for OSCC patients.
... A large number of external and environmental influences, including drug treatments under laboratory conditions, exert a promoting or inhibiting effect on the overall growth of cell populations [1][2][3][4], which is typically represented and visualized as biological growth curves [5][6][7][8][9][10]. For the assessment of positive or negative influences on these populations, a comparison of growth curves derived from treated and non-treated control groups can be performed in these cases [11,12], and-if significant changes between groups are detected-quantitative estimation of this effect can be performed. ...
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Heteroplasmy occurs when wild-type and mutant mitochondrial DNA (mtDNA) molecules co-exist in single cells¹. Heteroplasmy levels change dynamically in development, disease and ageing2,3, but it is unclear whether these shifts are caused by selection or drift, and whether they occur at the level of cells or intracellularly. Here we investigate heteroplasmy dynamics in dividing cells by combining precise mtDNA base editing (DdCBE)⁴ with a new method, SCI-LITE (single-cell combinatorial indexing leveraged to interrogate targeted expression), which tracks single-cell heteroplasmy with ultra-high throughput. We engineered cells to have synonymous or nonsynonymous complex I mtDNA mutations and found that cell populations in standard culture conditions purge nonsynonymous mtDNA variants, whereas synonymous variants are maintained. This suggests that selection dominates over simple drift in shaping population heteroplasmy. We simultaneously tracked single-cell mtDNA heteroplasmy and ancestry, and found that, although the population heteroplasmy shifts, the heteroplasmy of individual cell lineages remains stable, arguing that selection acts at the level of cell fitness in dividing cells. Using these insights, we show that we can force cells to accumulate high levels of truncating complex I mtDNA heteroplasmy by placing them in environments where loss of biochemical complex I activity has been reported to benefit cell fitness. We conclude that in dividing cells, a given nonsynonymous mtDNA heteroplasmy can be harmful, neutral or even beneficial to cell fitness, but that the ‘sign’ of the effect is wholly dependent on the environment.
It is difficult to show microbial growth kinetics online when they grow in complex matrices. We presented a novel strategy to address this challenge by developing a high-performance microbial growth analyzer (HPMGA), which employed a unique 32-channel capacitively coupled contactless conductivity detector as a sensing element and fixed with a CellStatz software. It was capable of online showing accurate and repeatable growth curves of well-dispersed and bad-dispersed microbes, whether they grew in homogeneous simple culture broth or heterogeneous complex matrices. Moreover, it could automatically report key growth kinetics parameters. In comparison to optical density (OD), plate counting and broth microdilution (BMD) methods, we demonstrated its practicability in five scenarios: 1) the illustration of the growth, growth rate, and acceleration curves of Escherichia coli (E. coli); 2) the antimicrobial susceptibility testing (AST) of Oxacillin against Staphylococcus aureus (S. aureus); 3) the determination of Ag nanoparticle toxicity on Providencia rettgeri (P. rettgeri); 4) the characterization of milk fermentation; and 5) the enumeration of viable pathogenic Vibrio in shrimp body. Results highlighted that the HPMGA method had the advantages of universality and effectivity. This technology would significantly facilitate the routine analysis of microbial growth in many fields (biology, medicine, clinic, life, food, environment, and ecology), paving an avenue for microbiologists to achieve research goals that have been inhibited for years due to a lack of practical analytical methods.
