Hello everybody!
I'm isolating and culturing HUVECs for a few months and I already charactirize them on FACS with CD 31, CD 144 positive and CD 45 negative. On my last characterization on my last isolation I had CD 31 and CD 144 very positive like before but I had like 98% of CD45 positive. I don't really explain that result. After isolation of HUVECs I culture them for a few days and when they are almost confluents I trypsinize them for FACS so I shouldn't have any other blood cells normally.
Can anyone help me please? What CD 45 + means?
Thank you very much and good day
Charles
Usually adherent cells are CD45- as you mention. What I can tell you from my experience is that dead cells have high CD45 FITC. It´s always advisable to run a FITC isotype with the same cells as well. I doubt they are CD45 +, you should repeat the staining, most probably there was a particular problem with those cells.
I agree with the above. Try not to use trypsin, which can affect many markers that are expressed on the surface.
And, use isotype control antibody for CD45 to identify the negative gate. I used to use CD45-APC, which has a strong background. Also, dead cells may also have auto-fluorescence.
However, I remember an old paper (very old) saying endothelial cells also express CD45.
I am also having CD45+ HUVEC cells. My CD45 antibody is conjugated to BB515 fluorophore, and I also ran isotype control. I still can't explain why.
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