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Validation of our modified MTS assay protocol. Prostate cancer cells (DU145) were plated onto all wells of 96 well plates. (a) 3 days after media change they were assayed by MTS as per manufacturer's instructions, our modified MTS protocol or MTT assay. Results are displayed as relative OD compared to cells in row 4 (*p < 0.05 by one way ANOVA and subsequent multiple comparison test, compared to row 4). (b) Prostate cancer cells were then plated in the 3rd and 4th row of a 96 well plate and the surrounding outside wells were left empty, filled with PBS, media alone or supplemented with 10% FBS. MTS assay was performed on the wells with cells after 3 days as per manufacturer's recommendations. Results displayed are means of row 3 and 4 together. (c) Media from wells in experiment (b) were measured by aspiration and weighing (*p < 0.05 by one way ANOVA and subsequent multiple comparison test, compared to media with FCS).
Source publication
The 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) calorimetric assay is replacing the traditional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as a fast, one-step assay of cell viability. We have observed that evaporation of the outer wells of a 96 well plate increases the...
Contexts in source publication
Context 1
... to addition of the MTS reagent (20 ll), the old culture media was removed and replaced with fresh media (100 ll). Figure 2a shows that with the modified protocol, there were no signifi- cant differences between the rows (p = 0.70). We further reasoned that MTT assay should not be susceptible to the evaporation problem because the insoluble formazan salt within the cells are solubilised in equal volumes of solvent prior to absorbency reading. ...
Context 2
... was a significant difference in absorbency between the wells surrounded with different solutions (p < 0.001). We found a 22% and 9.6% increase in absorbency within wells sur- rounded by empty wells and wells filled with PBS, respectively, compared to wells surrounded with media ( Figure 2b). There was no difference between wells surrounded by media alone or containing FCS. Correspondingly, there were also significant differences in the volume mea- sures between different wells (p < 0.001). ...
Context 3
... was no difference between wells surrounded by media alone or containing FCS. Correspondingly, there were also significant differences in the volume mea- sures between different wells (p < 0.001). There was a 17% and 4% decrease in volume between wells surrounded by empty and PBS filled wells, respectively, when compared to wells surrounded with media +/)FCS (Figure 2c). Based on these results, we suggest that if periods of culture, longer than 3 days without media change are re- quired or less resilient cells are cultured, than cells should only be plated in the 3rd and 4th row, with the outer rows surrounded by media containing wells. ...
Citations
... 11,14 Some aspects of CLD suitability of DWP have been studied, including oxygen and nutrient availability, mix and agitation efficiency, and evaporation control. [15][16][17][18][19] However, a few other key considerations, especially cross-contamination risk during vigorous shaking and cell transfer, have not been reported yet. ...
Cell line development (CLD) plays a crucial role in the manufacturing process development of therapeutic biologics. Most biologics are produced in Chinese hamster ovary (CHO) cell. Because of the nature of random transgene integration in CHO genome and CHO's inherent plasticity, stable CHO transfectants usually have a vast diversity in productivity, growth, and product quality. Thus, we often must resort to screening a large number of cell pools and clones to increase the probability of identifying the ideal production cell line, which is a very laborious and resource‐demanding process. Here we have developed a deep‐well plate (DWP) enabled high throughput (DEHT) CLD platform using 24‐well DWP (24DWP), liquid handler, and other automation components. This platform has capabilities covering the key steps of CLD including cell passaging, clone imaging and expansion, and fed‐batch production. We are the first to demonstrate the suitability of 24DWP for CLD by confirming minimal well‐to‐well and plate‐to‐plate variability and the absence of well‐to‐well cross contamination. We also demonstrated that growth, production, and product quality of 24DWP cultures were comparable to those of conventional shake flask cultures. The DEHT platform enables scientists to screen five times more cultures than the conventional CLD platform, thus significantly decreases the resources needed to identify an ideal production cell line for biologics manufacturing.
... It is reported that evaporation was observed from the outer wells of 96-well plate during incubation while using the MTS assay. The evaporation induced a 40%-52% increase in absorbency in the 1st and 2nd row as compared to the 3rd and 4th one of the 96-well plate while using MTS assay and gives false results [131][132][133]. ...
Cancer has become the silent killer in less-developed countries and the most significant cause of morbidity worldwide. The accessible and frequently used treatments include surgery, radiotherapy, chemotherapy, and immunotherapy. Chemotherapeutic drugs traditionally involve using plant-based medications either in the form of isolated compounds or as scaffolds for synthetic drugs. To launch a drug in the market, it has to pass through several intricate steps. The multidrug resistance in cancers calls for novel drug discovery and development. Every year anticancer potential of several plant-based compounds and extracts is reported but only a few advances to clinical trials. The false-positive or negative results impact the progress of the cell-based anticancer assays. There are several cell-based assays but the widely used include MTT, MTS, and XTT. In this article, we have discussed various pitfalls and workable solutions.
Graphical abstract
... It is important to note that wells on the edge of the plate were not used, as the authors observed an overestimation of nitrite concentrations (see supplementary Figure 2). Edge effects can contribute to assay variability and are usually associated with increased evaporation from outer wells after incubation for long periods of time [28]. To compare activity of the different MSC-sEV preparations, the authors decided to standardize the dose by loading the same number of particles (1 £ 10 9 particles/mL), as determined by nanoparticle tracking analysis. ...
Background aims
Owing to the lack of biological assays, determining the biological activity of extracellular vesicles has proven difficult. Here the authors standardized an in vitro assay to assess the anti-inflammatory activity of mesenchymal stromal cell-derived small extracellular vesicles (MSC-sEVs) based on their ability to prevent acquisition of the M1 phenotype in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Induction of tumor necrosis factor alpha, IL-1β, IL-6 and inducible nitric oxide synthase (iNOS) characterizes the M1 phenotype. Nitric oxide released by iNOS turns into nitrite, which can be easily quantitated in culture media by Griess reaction.
Methods
The authors first tested different assay conditions in 96-well plates, including two seeding densities (2 × 10⁴ cells/well and 4 × 10⁴ cells/well), four LPS doses (1 ng/mL, 10 ng/mL, 100 ng/mL and 1000 ng/mL) and two time points (16 h and 24 h), in order to determine the best set-up to accurately measure nitrite concentration as an index of M1 macrophage polarization.
Results
The authors found that seeding 2 × 10⁴ cells/well and stimulating with 10 ng/mL LPS for 16 h allowed the inhibition of nitrite production by 60% with the use of dexamethasone. Using these established conditions, the authors were able to test different MSC-sEV preparations and generate dose–response curves. Moreover, the authors fully analytically validated assay performance and fulfilled cross-validation against other M1 markers.
Conclusions
The authors standardized a quick, cheap and reproducible in vitro macrophage assay that allows for the evaluation and estimation of the anti-inflammatory activity of MSC-sEVs.
... The edge effect found in this study could be caused by differences in temperature across the plate, with lower temperatures in the center of the plate than in the edges, as has been described previously (5,7,15). Increased temperatures at the edges will cause more evaporation in outer wells and results in higher OD values (16,17). Also, wells located at the edges are not fully surrounded by other wells, which can affect the temperature in those wells (15), and temperature differences between filled wells (higher) and empty wells (lower) have also been described (5). ...
The diagnosis of Lyme neuroborreliosis (LNB) is based on neurological symptoms, cerebrospinal fluid (CSF) pleocytosis, and intrathecally produced Borrelia -specific antibodies. In most cases, the presence of intrathecally produced Borrelia -specific antibodies is determined by using an enzyme-linked immunosorbent assay (ELISA). The edge-effect is a known phenomenon in ELISAs and can negatively influence the assay reproducibility, repeatability, as well as index calculations of sample pairs which are tested in the same run.
... The cells were subsequently seeded in 96-well ultra-low attachment plates (Corning, Wiesbaden, Germany) at a density of 2000 cells/well. To avoid edge effects caused by greater evaporation in the circumferential wells of the 96-well plate, 16,17 only the inner 60 wells were used on each plate. The plates were centrifuged at 200Âg for 2 min and placed in a humified incubator at 37 C, 5% CO 2 . ...
3D cultures of primary human hepatocytes (PHH) are emerging as a more in vivo-like culture system than previously available hepatic models. This work describes the characterisation of drug metabolism in 3D PHH spheroids. Spheroids were formed from three different donors of PHH and the expression and activities of important cytochrome P450 enzymes (CYP1A2, 2B6, 2C9, 2D6, and 3A4) were maintained for up to 21 days after seeding. The activity of CYP2B6 and 3A4 decreased, while the activity of CYP2C9 and 2D6 increased over time (P < 0.05). For six test compounds, that are metabolised by multiple enzymes, intrinsic clearance (CLint) values were comparable to standard in vitro hepatic models and successfully predicted in vivo CLint within 3-fold from observed values for low clearance compounds. Remarkably, the metabolic turnover of these low clearance compounds was reproducibly measured using only 1–3 spheroids, each composed of 2000 cells. Importantly, metabolites identified in the spheroid cultures reproduced the major metabolites observed in vivo, both primary and secondary metabolites were captured. In summary, the 3D PHH spheroid model shows promise to be used in drug discovery projects to study drug metabolism, including unknown mechanisms, over an extended period of time.
... However, evaporation there was almost no evaporation from well 2 (glucose) and poor growth in well 3 (xylose). Evaporation was not expected to affect the maximum specific growth rate of strains which reached stationary phase within less than 80 h, but the effect was avoided in cultures of LF562, LF580, KF525 and LF458 by filling outer wells with water [82]. This reduced the number of replicates or conditions which were measured in a single plate. ...
Various marine fungi have been shown to produce interesting, bioactive compounds, but scaling up the production of these compounds can be challenging, particularly because little is generally known about how the producing organisms grow. Here we assessed the suitability of using 100-well BioScreen plates or 96-well plates incubated in a robot hotel to cultivate eight filamentous marine fungi, six sporulating and two non-sporulating, to obtain data on growth and substrate (glucose, xylose, galactose or glycerol) utilisation in a high throughput manner. All eight fungi grew in both cultivation systems, but growth was more variable and with more noise in the data in the Cytomat plate hotel than in the BioScreen. Specific growth rates between 0.01 (no added substrate) and 0.07 h⁻¹ were measured for strains growing in the BioScreen and between 0.01 and 0.27 h⁻¹ for strains in the plate hotel. Three strains, Dendryphiella salina LF304, Penicillium chrysogenum KF657 and Penicillium pinophilum LF458, consistently had higher specific growth rates on glucose and xylose in the plate hotel than in the BioScreen, but otherwise results were similar in the two systems. However, because of the noise in data from the plate hotel, the data obtained from it could only be used to distinguish between substrates which did or did not support growth, whereas data from BioScreen also provided information on substrate preference. Glucose was the preferred substrate for all strains, followed by xylose and galactose. Five strains also grew on glycerol. Therefore it was important to minimise the amount of glycerol introduced with the inoculum to avoid misinterpreting the results for growth on poor substrates. We concluded that both systems could provide physiological data with filamentous fungi, provided sufficient replicates are included in the measurements.
... In the present study, for determination the cell viability and cytotoxic effect of the test compounds, MTS and LDH methods were used, respectively. Using MTS method, the number of viable cells is determined in a very short time by a simple procedure (Patel et al. 2005). LDH assay is a means of measuring cytotoxicity following cellular membrane damage (Heeg et al. 1985). ...
Brain cancers are one of the most aggressive tumours in humans. Especially, gliomas are among the deadliest of human cancers and show high
resistance to chemotherapeutic agents. On the other hand, discovery of biologically effective non-synthetic biomaterials in treatments of different diseases, especially cancer, has continued to be one of the most popular research topics today. Therefore, we aimed to investigate biochemical, cytological and molecular genetic effects of napelline and talatisamine diterpenes in human U-87 MG glioma cells by using total antioxidant status and total oxidative status, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxphenyl)-2-(4-sulfophenyl)-2H-tetrozolium, inner salt and lactate dehydrogenase release assay and RT2 Prolifer PCR Arrays. Our results revealed that napelline and talatisamine exhibited cytotoxic effects at high doses. Napelline and talatisamine diterpenes increased apoptosis compared to control in U-87 MG cells. While napelline induced up-regulation of 50 and down-regulation of 13 genes, talatisamine induced up-regulation of 32 and down-regulation of 18 genes in U-87 MG cells. Napelline was shown to have a higher anticancer activity than talatisamine. We think that, napelline and talatisamine might be evaluated as potential chemotherapeutic agents for treatment of glioblastoma.
... Well effect. Microplate experiments are known to have well position and edge effects, where wells on the edges of the plate tend to give different results due to more rapid liquid evaporation [42][43][44][45] . Another potential source of bias is the well reading order, which starts at A1 to A12, then B1 to B12, until well H12 is reached. ...
Identification of allergenic IgE epitopes is instrumental for the development of novel diagnostic and prognostic methods in food allergy. In this work, we present the quantification and validation of a Bead-Based Epitope Assay (BBEA) that through multiplexing of epitopes and multiple sample processing enables completion of large experiments in a short period of time, using minimal quantities of patients’ blood. Peptides that are uniquely coupled to beads are incubated with serum or plasma samples, and after a secondary fluorophore-labeled antibody is added, the level of fluorescence is quantified with a Luminex reader. The signal is then normalized and converted to epitope-specific antibody binding values. We show that the effect of technical artifacts, i.e. well position or reading order, is minimal; and batch effects - different individual microplate runs - can be easily estimated and eliminated from the data. Epitope-specific antibody binding quantified with BBEA is highly reliable, reproducible and has greater sensitivity of epitope detection compared to peptide microarrays. IgE directed at allergenic epitopes is a sensitive biomarker of food allergy and can be used to predict allergy severity and phenotypes; and quantification of the relationship between epitope-specific IgE and IgG4 can further improve our understanding of the immune mechanisms behind allergic sensitization.
... 8,9 A longer incubation of plates over 24 h is susceptible to faster evaporation of the medium from peripheral wells, which results in inconsistent readouts. 10,11 Furthermore, small culture volumes in MPs are more prone to evaporation-induced osmolality shifts in the medium that critically affect the growth and metabolic activities of cells. 9,10,12,13 Sustainable improvements in culture conditions that prevent uneven loss of the medium decrease well-to-well variability in assay readouts. ...
... A standard practice in MP-based assays is to exclude the wells in the periphery of plates to avoid bias in data analysis from edge effect. 11 This results in the loss of 20% of the MP working area, which may potentially increase the overall cost of consumables in the long run. In our study, we show that a combination of MicroClime lid and SteriStore incubator results in low spheroid variability in outer wells of 384WPs, making it possible to use the entire plate. ...
Spheroid cultures of cancer cells reproduce the spatial dimension–induced in vivo tumor traits more effectively than the conventional two-dimensional cell cultures. With growing interest in spheroids for high-throughput screening (HTS) assays, there is an increasing demand for cost-effective miniaturization of reproducible spheroids in microtiter plates (MPs). However, well-to-well variability in spheroid size, shape, and growth is a frequently encountered problem with almost every culture method that has prevented the transfer of spheroids to the HTS platform. This variability partly arises due to increased susceptibility of MPs to edge effects and evaporation-induced changes in the growth of spheroids. In this study, we examined the effect of evaporation on the reproducibility of spheroids of tumor and nontumor cell lines in 384-well plates, and show that culture conditions that prevent evaporation-induced medium loss result in the formation of uniform spheroids across the plate. Additionally, we also present a few technical improvements to increase the scalability of the liquid-overlay spheroid culturing technique in MPs, together with a simple software routine for the quantification of spheroid size. We believe that these cost-effective improvements will aid in further improvement of spheroid cultures for HTS drug discovery.
... However, they are not applicable for all measuring systems. Therefore, it is common laboratory practice to abandon the outer MTP wells in HTS to minimize the experimental noise [16,25]. However, this practice rather bypasses than solves the edge effect and impairs the idea of high-throughput measurements by reducing the efficiency and capacity of screening and development methods [9]. ...
... Evaporation is assumed to cause these temperature variations within MTPs [10,25], and, thereby, also variations of enzyme kinetics [28]. Plate sealing foils are often used to minimize evaporation and, thereby, edge effects [2,10,12,22]. ...
Microtiter plates (MTP) and automatized techniques are increasingly applied in the field of biotechnology. However, the susceptibility of MTPs to edge effects such as thermal gradients can lead to high variation of measured enzyme activities. In an effort to enhance experimental reliability, to quantify, and to minimize instrument-caused deviations in enzyme kinetics between two MTP-readers, we comprehensively quantified temperature distribution in 96-well MTPs. We demonstrated the robust application of the absorbance dye cresol red as easily applicable temperature indicator in cuvettes and MTPs and determined its accuracy to ±0.16 °C. We then quantified temperature distributions in 96-well MTPs revealing temperature deviations over single MTP of up to 2.2 °C and different patterns in two commercial devices (BioTek Synergy 4 and Synergy Mx). The obtained liquid temperature was shown to be substantially controlled by evaporation. The temperature-induced enzyme activity variation within MTPs amounted to about 20 %. Activity deviations between MTPs and to those in cuvettes were determined to 40 % due to deviations from the set temperature in MTPs. In conclusion, we propose a better control of experimental conditions in MTPs or alternative experimental systems for reliable determination of kinetic parameters for bioprocess development.
