SRB assay for measuring target cell killing
Diana Rose E Ranoa
Disclaimer
Protocol adapted from:
Vichai V, Kirtikara K. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nature Protocols 2006 ;1(3):1112-6 doi 10.1038/nprot.2006.179.
Abstract
The Sulforhodamine B colorimetric assay was used to measure effector CAR T cell killing of their target tumor cells.
Steps
Perform co-culture experiment of activated CAR T cells and target tumor cells at various tumor:effector ratio in a 96-well plate for 24 hours at 37'C, 5% CO2.
Remove cell culture supernatant 24 hours after co-culture assay.
Using a multichannel pipet, fix adherent tumor cells on the 96-well plate with 100uL cold 10% (w/v) tricholoroacetic acid (TCA) to each well, and incubate at 4'C for 1hr.
Wash four times by dipping entire plate in a basin with slow-running tap water. Tap plates on paper towels to remove excess water, and allow to dry at room temperature.
Add 100uL of 0.057% (w/v) Sulforhodamine B (SRB) solution to each well and incubate at room temperature for 30 minutes.
Immediately wash plates four times in a basin containing 1% (v/v) acetic acid to remove unbound dye.
After the plates have dried completely, add 200uL of 10mM Tris base solution (pH 10.5) to each well and incubate for 5 minutes with shaking.
Read plates in a microplate reader at A510nm.
Calculate the percentage of cell growth inhibition using the formula below:
% cells killed = 100 - [mean OD(co-cultured sample)/mean OD(tumor cell only)]x100
