I did an SRB (sulforhodamine B) test to see the effect of an inhibitor on HCT116 and HT29 colorectal cancer cells. I seeded the cells and incubated for 1 day. After 24 hours I gave the inhibitor and it was incubated for 48 hours. After 48 hours, when performing SRB tests with HCT116 and HT29 cells, TCA (trichloroacetic acid) remains in the wells including the blank wells and is stained with SRB dye. It leads to the wrong result. We think that TCA fixes the proteins in the serum (FBS). Could it be caused by something else? Why do you think it happens?
I attached a blank photo as a image.
Hello Senanur Malcanlı
Yes, TCA does fix the proteins in serum.
You could do the following.
1.The signal-to-noise ratio is higher when culture medium is supplemented with 5% FBS rather than 10% FBS.
2. You could centrifuge the microplate and subsequently remove the supernatant prior to fixation.
3. You could use a microplate-multiwash device to aspirate the medium before fixation. The tips of this device aspirate the medium in a consistent manner, leaving only a minimal amount of culture medium approximately which will produce a negligible background effect although it adds one more step to the already multi-step SRB assay. Flicking and tapping of microplates or aspiration of medium by multichannel pipettes can result in the loss of an unpredictable number of cells.
Please refer to the paper attached for more information on the optimization of the sulforhodamine B colorimetric assay.
Article Optimization of the sulforhodamine B colorimetric assay
Best.
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