I am trying to quantify the percentage of retinal ganglion cells I have in my cell population by surface staining of CD90 using flow cytometry.
The protocol I follow have a blocking step with 2% goat serum (the host species of the secondary antibody) and I use a primary antibody for CD90 and then, a secondary one. The antibodies are:
- Invitrogen CD90 Monoclonal Antibody (F15-42-1), Invitrogen™. Ref: 15215939
- Proteintech IgG(H+L) Goat anti-Mouse, FITC, Secondary Antibody, Proteintech. Ref: 17660327
I have tried a lot of different dilutions for the antibodies, even at lower concentration that the range recommended by the suppliers; and I incubate them for only 7 minutes (the supplier recommends 30 minutes for each one).
My problem is that even lowering the concentration and the incubation time, all the cells stain positive for CD90 and it is unspecific detection because I have checked by other methods and the percentage of positive cells should be very low.
I do not know if maybe the blocking step is failing for some reason, or if it's a problem of the antibody that recognizes proteins in an unspecific manner.........
Thank you very much.
Dear Urs Mörbe,
Thank you for your answer. Yes, I am analyzing human retinal ganglion cells that have been obtained by differentiating induced pluripotent stem cells, so I presumptly have a mixed population containing retinal ganglion cells and other retinal cells, as well. I have previously checked by quantitative PCR the expression of CD90 and it expresses in the cells but its expression is relatively "low". So, I do expect to obtain small percentage of CD90-positive cells.
The antibody I am using gives strong signal but maybe it is causing unespecific staining, and the problem is that I don't know if it's a problem of the antibody or if it is something in the technique (I don't think so, by the way...). I use only this antibody, it's not used in combination with another one.
I would consider using the antibody you are recommending... and maybe it would be interesting to have some kind of control cells that do not express that antigen, to discriminate unspecific staining of the antibody.
Best regards,
Marta