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 large number of miRNA mimics or compounds in a single plate (> 60 per plate, 6 replicates) using inexpensive equipment and reagents.
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