This chapter is an introductory overview of the most commonly used assay methods to estimate the number of viable cells in multi-well plates. This chapter describes assays where data are recorded using a plate-reader; it does not cover assay methods designed for flow cytometry or high content imaging. The assay methods covered include the use of different classes of colorimetric tetrazolium reagents, resazurin reduction and protease substrates generating a fluorescent signal, the luminogenic ATP assay, and a novel real-time assay to monitor live cells for days in culture. The assays described are based on measurement of a marker activity associated with viable cell number. These assays are used for measuring the results of cell proliferation, testing for cytotoxic effects of compounds, and for multiplexing as an internal control to determine viable cell number during other cell-based assays.
Cell-based assays are often used for screening collections of compounds to determine if the test molecules have effects on cell proliferation or show direct cytotoxic effects that eventually lead to cell death. Cell-based assays also are widely used for measuring receptor binding and a variety of signal transduction events that may involve the expression of genetic reporters, trafficking of cellular components, or monitoring organelle function. Regardless of the type of cell-based assay being used, it is important to know how many viable cells are remaining at the end of the experiment. There are a variety of assay methods that can be used to estimate the number of viable eukaryotic cells. This chapter will provide an overview of some of the major methods used in multi-well formats where data are recorded using a plate reader. The methods described include: tetrazolium reduction, resazurin reduction, protease markers, and ATP detection. Methods for flow cytometry and high content imaging may be covered in different chapters in the future.
Lymphocyte Proliferation Assay Pdf Download
The MTT assay was developed as a non-radioactive alternative to tritiated thymidine incorporation into DNA for measuring cell proliferation (1). In many experimental situations, the MTT assay can directly substitute for the tritiated thymidine incorporation assay (Figure 3).
However, it is worth noting that MTT reduction is a marker reflecting viable cell metabolism and not specifically cell proliferation. Tetrazolium reduction assays are often erroneously described as measuring cell proliferation without the use of proper controls to confirm effects on metabolism (10).
Cell proliferation was assessed using MTT assay. Cells were cultured in 96-well plates and the cell numbers were quantified using a coulter counter (Coulter Electronics, Inc., Hialeah, FL). Each well contained 2 104cells in a total volume of 200 μL. The plate included blank wells (0 cells/mL), control wells (2 104cells/0.2 Ml, untreated group), control wells with DMSO (no cells), control wells treated with SR140333 (10-9M-10-5M), control wells treated with SMSP (10-10M-10-6M) and control wells treated with SMSP (most effective concentration) combined with different concentrations of SR140333 (10-9M-10-5M). Drugs were added on day 3 (at exponential phase) and the assay was performed after 24 hours. For the proliferation assay, 20 μL MTT was added in each well. After 4 hour at 37C supernatant was removed and 100 μL DMSO was added in each well. The optical density (OD) was detected in the microplate reader at 570 nm wavelength (Biotech Instruments, New York, USA). Each experimental condition (blank wells, control wells, and control wells treated with drugs) was assayed in duplicate and each study was repeated on at least three separate occasions. Representative data from each experiment are shown in this article.
Statistical analysis was performed with SPSS 10.0 statistical software for Microsoft Windows. Values of proliferation assay and growth study were expressed as means SD. Data from the proliferation assay were analyzed using one-way ANOVA. The homogeneity of the variance was tested using the Levene test. If the variances were homogeneous, Fischer's least significant difference procedure for multiple comparisons with Bonferroni adjustment and Dunnett t tests were used. For data sets with non-homogeneous variances, the ANOVA test with T3 Dunnett post hoc analysis was applied. Data from growth study were analyzed using Dunnett t tests. We only considered the variances among different treating factors at the same day. The criterion for significance was p
This protocol provides an overview of the IncuCyte Cell Count Proliferation Assay methodology. It is compatible with the IncuCyte live-cell analysis system and enables real-time cell counting using your choice of cells and treatments. The IncuCyte NucLight range of live cell labelling reagents are used to fluorescently label the nuclei of living cells without perturbing cell function or biology. Transient or stable expression strategies are supported using either the IncuCyte NucLight BacMam 3.0 or NucLight Lentivirus range of reagents. In addition, the highly flexible assay format can be combined with our range of IncuCyte Cytotox Reagents or the IncuCyte Caspase 3/7 reagent for multiplexed measurements of cytotoxicity and apoptosis alongside proliferation in the same well.
B cell proliferation assays were performed as described to investigate if the impact of MMF on B cell activation and generation of ASCs that we postulated in patients with lupus, was indirect or direct. The MPA concentration that was used in all functional assays had been chosen considering previous work, and the approximate plasma concentration reached by daily intake of 2-3 g MMF [37].
Proliferation assays of purified CD27-IgD+ antigen-naïve and CD27+ memory B cell subsets from the peripheral blood of four healthy blood donors were performed. All individuals showed a comparable pattern of proliferation and a representative example is shown (Figure 4). In all assays MPA was able to abolish B cell proliferation and differentiation of ASCs completely, no matter if it was caused by TLR-mediated stimulation using CpG, or by CD40-antibodies in combination with IL-21, suggesting that the impact of MMF on B-cell activation and proliferation is not only related to impaired T cell help. In contrast to calcineurin inhibitors [38], MMF inhibits B cell activation and differentiation of ASCs directly.
MPA abolishes B cell proliferation and plasma cell formation. Mycophenolic acid (MPA) completely abolishes CPG- or anti-CD40 +IL21-induced proliferation of CD27-antigen-naïve B cells (A) or CD27+ memory B cells (B). Results of a representative B cell proliferation assay of a healthy donor are shown. CD27-IgD+ B cells (A) or CD27+ B cells (B) were isolated, labeled with carboxy-fluorescein-succinimidyl ester (CFSE) and stimulated for 3 to 4 days as indicated. Gated on live cells, plasmablast gates are shown. Dividing-B cells lose CFSE with any cell division.
Treatment of lupus patients with MMF causes an improvement of symptoms as well as normalization of paraclinical aberrations, such as increased peripheral plasmablast counts or hypergammaglobulinemia. Its preferential effect on B cell activation, proliferation and plasma cell generation is most welcome in patients with SLE and seems to be associated with high therapeutic efficacy combined with favorable safety. While controlled clinical trials have recently demonstrated the latter, this study focused on paraclinical aspects. Combining observational data and results obtained by performing selected functional assays it suggests modes of action that are especially advantageous in lupus, a disease characterized by enhanced B cell activation and proliferation, as well as plasma cell expansion and autoantibody secretion.
Until recently, the impact of MMF on human B cell subsets has been neglected, because T lymphocytes enjoyed unshared attention as key players of allograft rejection. A study published a few years ago addressed the in vitro effect of MPA on purified total human B cells and described an inhibition of CD40-induced proliferation [40]. Another study investigated the effect of starting MMF, on peripheral B and T cell activation markers in 10 patients with SLE [41] and found a decrease of CD38++CD19+ B cells in most patients.
In the poultry industry, quantitative analysis of chicken T cell proliferation is important in many biological applications such as drug screening, vaccine production, and cytotoxicity assessment. Several assays have been established to evaluate this immunological response in chicken cells. However, these assays have some disadvantages including use of radioactive labels ([3H]-Thymidine assay), necessity of DNA denaturation or digestion (BrdU incorporation assay), lack of sensitivity and underestimation of anti-proliferative effects (MTT assay), and modulation of activation molecules and cell viability reduction (CFSE assay). Overcoming these limitations, the EdU proliferation assay is sensitive and advantageous compared to [3H]-Thymidine radioactive labels in studies on cell proliferation in vitro and allows simultaneous identification of T cell populations. However, this assay has not been established using primary chicken cells to evaluate T cell proliferation by flow cytometry.
Here, we established an assay to evaluate the proliferation of primary chicken splenocytes based on the incorporation of a thymidine analog (EdU) and a click reaction with a fluorescent azide, detected by a flow cytometer. We also established a protocol that combines EdU incorporation and immunostaining to detect CD4+ and CD8+ proliferating T cells. By inducing cell proliferation with increasing concentrations of a mitogen (Concanavalin A), we observed a linear increase in EdU positive cells, indicating that our protocol does not present any deficiency in the quantity and quality of reagents that were used to perform the click reaction. 2ff7e9595c
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