I am trying to set up a protocol to study circulating endothelial cells by flow cytometry. First approach was to identify CD45-CD146+ population and analyze expression of cell adhesion molecules on them. It worked well: I have identified about 0.4% of CEC out of all mononuclear cells after myocardial infarction which decreased up to 0.06% on day 3. But then I wanted to analyze expression of eNOS in these cells and had to apply fix/perm protocol: fixed cells after surface staining; permeabilzed them (both buffers are from BD, and worked well for staining of intracellular cytokines); then stained eNOS. The problem is that I have found no CD146+ in this sample at all. (The sample was from the same patient and the same number of events was collected as for only surface markers staining). A few CD45+CD146- cells were positive for e. May be anyone knows, what may be the problem with my protocol?
Without knowing how harsh your fixing solution is (4% PFA?), perhaps you are quenching the surface marker fluorescence after fixing. A gentler fixing protocol (lower % PFA), or incubating with an unconjugated primary antibody, then fix/permeabilise, then staining with secondary antibody-conjugated fluorophore could help. Hope these suggestions help, or inspire better ideas.
Thank you, Adrian. Unfortunately I am limited to the spectrum of reagents I possess at the moment. But PFA concentration is 1% in fixing buffer. I will consider your suggestions for the future.
Totally understand Irina Kologrivova, I hope you can think of something.
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