Please help... - (Mar/24/2014 )

Hello everyone, 

I have a problem with the staining of my cells. I am isolating intestinal myofibroblasts (iMF) from mouse large and small bowel and to address the number of these cells after isolation and during cultivation, after passaging, I perform flow cytometry analysis. I use anti-aSMA, anti-vimentin and anti-CD90 to detect iMF.

aSMA and vimentin stain nicely, but anti-CD90 does not work properly. It has been reported that iMF are positive for CD90, FACS blots in several publications show more than 80-90% of aSMA+ iMF-like cells being CD90 positive. Now in my case I see only 20-25% of aSMA+ iMF-like cells being positive for CD90. 

I tested the antibody on thymoid cells (isolated from mouse thymus) and it stains nicely (as expected). Accutase treatment, medium, collagenase and dispase, used for passaging, cultivation and in isolation from the IG lamina propria respectively, have no effet on CD90 signal on the thymoid cells.

Has anyone an idea what the reason for this discrepancy might be? Why does the anti-CD90 antibody not work with iMF, but thymoid cells show expected results?

Thank you very much for a feedback!

1. When it worked on the thymoid cells, did you have the same co-stainings as with your iMF cells or did you just check the single CD90 staining on thymoid cells ? Asking to exclude a possible interference with the other two stainings.

2. Did you use the same conditions in thymoid and iMF cells with respect to staining procedure (fixation, simultanous vs. sequential co-staining) ?

3. Did you check approx. CD90 expression of your iMF with a different method (such as WB) and a positive control, if available ? To exclude that you iMF cells for whatever reason just really express a lower amount than expected.

Hmm... we did not perform co-staining with the other antibodies used (that might be the issue). However we used the exact same staining protocol and  checked the CD90 expression with WB, which showed a strong signal. 

Thanks for the fast answer, tbh I did not know that antibody interference may cause such bias.