May 24, 2023
Version 1
SRB assay for measuring target cell killing V.1
- 1University of Illinois at Urbana-Champaign
Protocol Citation: Diana Rose E Ranoa 2023. SRB assay for measuring target cell killing. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwjk7dlmk/v1Version created by Diana Rose E Ranoa
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 27, 2022
Last Modified: May 24, 2023
Protocol Integer ID: 71933
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.
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