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

1. SRB Assay for Pancreatic Carcinoma Cells

1. Dissolve SRB reagent in 1% acetic acid at final concentration of 0.4% (w/v).
2. Dissolve trichloroacetic acid (TCA) in ultrapure water at a final concentration of 50% (w/v).
3. Dissolve Tris(hydroxymethyl)-aminomethane in ultrapure water at a final concentration of 10 mM.
4. Prepare a separate 96-well flat-bottom "Day 0" control plate to ensure more accurate estimation of growth inhibition: Seed 6 wells with cells growing in exponential phase in 100 µL medium at appropriate seeding concentration and add 100 µL medium only to wells corresponding to blanks. Incubate overnight at 37 °C with 5% CO₂ to ensure proper adhesion of the cells to the plate. Then, add 100 µL medium to all the wells and proceed to steps 1.8–1.16 of this section.
5. Prepare a 96-well flat-bottom experimental plate: Seed cells growing in exponential phase in triplicate in 96-well flat-bottom plates at the appropriate density in 100 µL of medium by using a multichannel pipette.
   NOTE: In order to determine optimal starting cell concentrations, it is recommended to assess the growth profile of each cell line in a 96-well plate by seeding cells at several concentrations and measuring it daily for at least 4 days. Choose a seeding concentration that prevents overgrowth of cells after 72 h, as this will influence the experiment by saturating the OD values. For Panc-1 and Panc-1R cells, the optimal seeding concentration is 8000 cells/well.
6. Add 100 µL medium to medium-only wells and incubate overnight at 37 °C with 5% CO₂ to ensure proper adhesion of the cells to the plate.
7. Prepare a drug dilution range of gemcitabine between 1 µM and 10 nM for Panc-1 and 1 mM and 100 nM for Panc-1R cells. Add 100 µL 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. In addition, add 100 µL medium to the medium-only wells and the control cells. Incubate at 37 °C with 5% CO₂ for 72 h.
8. Add 25 µL of cold TCA solution to the wells by using a multichannel pipette and incubate the plates for at least 60 min at 4 °C to precipitate and fix the proteins at the bottom of the wells.
9. Empty the plate by removing the medium and dry briefly on a tissue.
10. Wash 5 times with tap water, then empty the plate and let dry at room temperature.
11. Add 50 µL SRB solution per well by using a repeat-pipette and stain for 15 min at room temperature.
12. Empty the plate by removing the SRB stain.
13. Wash 4 times with 1% acetic acid, then empty the plate and let it dry at room temperature.
14. Add 150 µL Tris solution per well by using a multichannel pipette and mix for 3 min on a plate-shaker up to a maximum of 900 shakes/min.
15. Read the optical density at 540 nm (or 492 nm if the OD values are too high).
16. Analyze the data.

2. Data Analysis for SRB Assay

1. Calculate the OD values of cells at "Day 0" using the following formula:
   OD_{Day 0} = OD_{control cells} – Average OD_{blank wells}
2. Calculate the percentage of surviving cells for each drug concentration according to the following formula:
   % Treated cells = Average [OD_{treated cells} - Average OD_{blank wells} - OD_{Day 0}]/[OD_{control cells} - Average OD_{blank wells} – OD_Day0]*100
3. Plot the dose-response curve (drug concentration vs. growth inhibition in %).
4. Calculate the concentration of the drug that inhibits the growth of cells by 50% (IC₅₀) using the dose-response curve.