Overview
This video describes the high throughput colorimetric screening assay, sulforhodamine B assay, to measure drug-induced cytotoxicity in cancer cells. The assay determines the drug resistance by measuring the cell density based on the cellular protein content.
Protocol
1. SRB Assay for Pancreatic Carcinoma Cells
- Dissolve SRB reagent in 1% acetic acid at final concentration of 0.4% (w/v).
- Dissolve trichloroacetic acid (TCA) in ultrapure water at a final concentration of 50% (w/v).
- Dissolve Tris(hydroxymethyl)-aminomethane in ultrapure water at a final concentration of 10 mM.
- 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.
- 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. - 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.
- 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.
- 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.
- Empty the plate by removing the medium and dry briefly on a tissue.
- Wash 5 times with tap water, then empty the plate and let dry at room temperature.
- Add 50 µL SRB solution per well by using a repeat-pipette and stain for 15 min at room temperature.
- Empty the plate by removing the SRB stain.
- Wash 4 times with 1% acetic acid, then empty the plate and let it dry at room temperature.
- 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.
- Read the optical density at 540 nm (or 492 nm if the OD values are too high).
- Analyze the data.
2. Data Analysis for SRB Assay
- Calculate the OD values of cells at "Day 0" using the following formula:
ODDay 0 = ODcontrol cells - Average ODblank wells - Calculate the percentage of surviving cells for each drug concentration according to the following formula:
% Treated cells = Average [ODtreated cells - Average ODblank wells - ODDay 0]/[ODcontrol cells - Average ODblank wells - ODDay0]*100 - Plot the dose-response curve (drug concentration vs. growth inhibition in %).
- Calculate the concentration of the drug that inhibits the growth of cells by 50% (IC50) using the dose-response curve.
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Materials
Name | Company | Catalog Number | Comments |
Sulforhodamine B | Sigma-Aldrich | 230162 | |
Trichloroacetic acid | Sigma-Aldrich | 251399 | |
Panc-1 | ATCC, Manassas, VA, USA | ATCC CRL-1469 | |
Tris(hydroxymethyl)-aminomethane | Sigma Aldrich | 252859 | |
Greiner CELLSTAR 96 well plates | Greiner/Sigma | M0812-100EA | |
Anthos-Elisa-reader 2001 | Labtec, Heerhugowaard, Netherlands | UV-Vis 96-well plate spectrophotometer | |
RPMI-1640 | Gibco, Carlsbad, CA, USA | 11875093 | |
Greiner CELLSTAR 96 well plates | Greiner/Sigma | M0812-100EA | |
Fetal bovine and calf serum | Greiner Bio-One, Frickenhausen, Germany | 758093 | |
penicillin G streptomycin sulphate | Gibco, Carlsbad, CA, USA | 15140122 |