I am running and experiment where I use cryopreserved PBMCs as the following:
1) thawing and incubation on RPMI-1640 in CO2 incubator overnight
2) activation with IFN-alpha for 15 min
3) fixation with BD fix/lyse buffer (10 min)
4) surface antigen staining
5) permeabilization with BD perm III buffer (30 min on ice)
6) intracellular antigen staining
When i run flow cytometry I get this image on FSC and SSC. I can't identify which population is lymphocytes as i see CD3+ in both population 2 and 3.
It iw true you might need experience to read the flow cytometry result, bit maybe say it could depend on both. Either you make sure your standard ( which could subtract the errors from controls) or from the properties of the lymphocytes. Size, density, granules, nucleus. You might see, but maybe is it "determinated"?. Maybe sqy it is a low experimental result.
As seen from the flow panel, cluster 1 is debris, 2 is lymphocytes, 3 is monocytes and 4 is SSC+ granulocytes.
The Lymphocytes are the most abundant population contributing more than 70% of the total cell population while monocytes make up a significantly smaller portion of the human PBMC. However, that not the same in the panel you show. Looks like cluster 3 is CD3+CD14+ cells consist of T cells bound to monocytes. Filtering out the doublets may help you to see right cell populations in right proportions. But, there comes a question that whether these PBMC's are from healthy donors? as it was recently shown that circulating T-cell-monocyte complexes are markers of immune perturbations such as infection or immunization, reflecting expected polarization of immune responses. If this is true, you may end up seeing CD3-CD14+, CD3+CD14mid, CD3+CD14hi populations within cluster 3. Similarly, CD3+CD14mid cells can also be expected within the lymphocyte gate.
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