I have a question about a picture I took with the confocal microscope. I isolated PBMCs from human blood using ficoll. I centrifuge it 15 minutes at 800xg without a brake.
After isolating the PBMC layer and washing with PBS I make cytospin samples and perform an immunofluorescent staining with y-H2AX, 53BP1 and dapi.
On some of the pictures I see these small green dots. I was wondering what this is, is it possible that these are red blood cells? or something else?
I think those are platelets, I have a similar problem. They seems to have some autofluorescence in the green channel and look very "sticky" for secondary antibodies and other things. You can have a better idea if it is erythrocytes or platelets just looking at the slide with phase contrast bright field microscope. Gerson
The green dots are platelets. Although Ficoll can isolate red blood cells from mononuclear cells, it cannot remove platelets. The best way to remove these platelets is MACS. If you only look at lymphocytes or monocytes, a simple way is to extract cells again with Percoll (a large number of protocols online), which can remove the platelets efficiently (but may still have a few left). Good luck!
I would exclude erythrocytes. The presence of platelets is a reasonable answer for your question. You could try the cell separation direct using Percoll gradients as suggested by Ruoyu Ma.
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