Hi,
I am experiencing an absurd problem while doing FACS. While staining OVCAR-3 cells with CD90 marker (for titration), i found that antibody washes away after centrifugation (which I guess means the antibody is not being strongly bound).
I stained cells, and then without washing around 100% of cells were positive with CD90, which is not possible as it is stem cell marker (Normally expression is supposed to be near 30%). However, upon washing with FACS buffer, all of the antibody washed out, giving 0% positive cells.
FACS buffer I used was 10% FBS in PBS with 0.5mM EDTA. Cells are live and stained for 45 minutes, before doing FACS.
Any help will be appreciated.
My interpretation is that none of your cells are CD90 positive. Add a positive control cell line to your experiment, if you have one available. This will give you more confidence in your negative result.
thanks for the help. Perhaps they have lost the expression, I will for sure check with stem cells.
I agree with Adam. It looks like your cells are not expressing CD90 at all. Try confirming it by Western blot or by QPCR.
Yes, for sure it was the same case. I tried with MSC-BM as they are stem cells and express CD90. it went good with them. perhaps my cancer cell line has lost the expression.
thanks for the help.
- Badges
- Science topic
- Similar topics
- Engineering
- Materials Engineering
- Powders