Sulforhodamine B Assay: A Sensitive Assay to Measure Drug Resistance Via Determination of Cellular Protein Content in Cancer Cells
Transcript
Sulforhodamine B or SRB assay allows cell density determination based on the measurement of cellular protein content. To begin the assay, pipette a suspension of desired drug-sensitive parental cancer cells and their drug-resistant variant into wells of a multi-well plate.
Incubate to allow the cells to adhere to the plate. Treat both the cell types with a range of increasing concentrations of the specific drug and incubate.
In culture, the drug-sensitive cancer cells are susceptible to the drug molecules and undergo cell death even at low concentrations. In contrast, the drug-resistant cancer cells withstand the effect of drug molecules and survive and proliferate even at higher drug concentrations.
Next, add an appropriate concentration of trichloroacetic acid or TCA to the culture. TCA – a mild acid – permeabilizes the cells and disrupts the electrostatic interactions and native protein conformations, leading to protein denaturation.
Incubate with Sulforhodamine B solution. Under mild acidic conditions, sulforhodamine B – a protein-binding dye – binds stoichiometrically to basic amino acid residues of proteins.
Rinse with acetic acid to remove any unbound dye. Now, treat the sample with a Tris base solution. The basic environment dissociates the dye molecules from the amino acid residues, releasing them into solution.
Finally, use a microplate reader to measure the released dye molecules. The wells containing drug-resistant cancer cells show higher color intensity, indicating increased cell density compared to the drug-sensitive variant cells.
