Abstract
The SRB assay has been used since its development in 1990 (Skehan et al., 1990) to inexpensively conduct various screening assays to investigate cytotoxicity in cell based studies (Vichai and Kirtikara, 2006). This method relies on the property of SRB, which binds stoichiometrically to proteins under mild acidic conditions and then can be extracted using basic conditions; thus, the amount of bound dye can be used as a proxy for cell mass, which can then be extrapolated to measure cell proliferation.The protocol can be divided into four main steps: preparation of treatment, incubation of cells with treatment of choice, cell fixation and SRB staining, and absorbance measurement. This assay is limited to manual or semiautomatic screening, and can be used in an efficient and sensitive manner to test chemotherapeutic drugs or small molecules in adherent cells. It also has applications in evaluating the effects of gene expression modulation (knockdown, gene expression upregulation), as well as to study the effects of miRNA replacement on cell proliferation (Kasinski et al., 2015).
Background
The SRB assay has been widely used to investigate cytotoxicity in cell based studies and it is the method of choice for high cost-effective screenings (Vichai and Kirtikara, 2006). Since this method does not rely on measuring metabolic activity [e.g., 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT], the steps required to optimize the protocol for a specific cell line are substantially simplified. The protocol described here has been optimized for medium throughput screening of miRNAs with tumor suppressive properties in adherent lung cancer cells in 96-well format and 384-well format (Kasinski et al., 2015). Particularly the SRB assay in 384-well format offers the advantage of screening a large number of miRNA mimics or compounds in a single plate (> 60 per plate, 6 replicates) using inexpensive equipment and reagents.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Subtract background absorbance from all wells. Calculate the percentage of cell-growth inhibition normalizing treatments to miRNA negative control using the following formula: % cell growth = Absorbance sample/Absorbance negative control or untreated x 100 (Figure 2B) % growth inhibition = 100 - % cell growth SRB assay results can only be used if the data falls within the linearity limit of the assay. We recommend performing a cell number titration experiment to determine the linear dynamic range of the assay with the cell line used (Figure 2A). Generally, O.D. of > 2 are not within the linear range of the assay. Statistical tests for this type of assay can include t-tests for two group comparisons or ANOVA for multiple group comparisons. Figure 2. Sulforhodamine B colorimetric assay in cell culture to analyze cell proliferation. A. Cell number titration (H441) using the SRB assay 384-well format, n = 6, error bars = SD. B. miR-34a dose response analysis in lung cancer cell lines H441 and H358 using SRB 96-well format, n = 3.
Notes
Acknowledgments
The protocol presented herein was adapted from Kasinski et al. (2015). This work was supported by an NIH grant (R00CA178091 and P30CA023168).
References
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