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