Sulforhodamine B Assay: A Sensitive Assay to Measure Drug Resistance Via Determination of Cellular Protein Content in Cancer Cells

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.

Prepare a 96-well flat-bottom experimental plate. Seed pancreatic carcinoma cells growing in exponential phase in triplicate in 96-well flat-bottom plates at the appropriate density in 100 microliters of medium by using a multichannel pipette.

Add 100 microliters of medium to medium-only wells and incubate overnight at 37 degrees Celsius with 5% carbon dioxide to ensure proper adhesion of the cells to the plate. Add 100 microliters from each dilution into an appropriate well of the 96-well plate by using a multichannel pipette. Make sure to have each concentration in triplicate. Incubate at 37 degrees Celsius with 5% carbon dioxide for 72 hours.

Next, add 25 microliters of cold TCA solution to the wells by using a multichannel pipette. Incubate the plates for at least 60 minutes at 4 degrees Celsius to precipitate and fix the proteins at the bottom of the wells.

Empty the plate by removing the medium and dry briefly on a tissue. Wash five times with tap water before emptying the plate and drying at room temperature. Add 50 microliters of SRB solution per well by using a repetitive pipette and stain for 15 minutes at room temperature.

Then, empty the plate by removing the SRB stain. After washing the plate four times with 1% acetic acid, empty the plate and let it dry at room temperature. Add 150 microliters of Tris solution per well by using a multichannel pipette and mix for 3 minutes on a plate shaker up to a maximum of 900 shakes per minute. Read the optical density at 540 nanometers.