I'm currently developing a human flow panel for peripheral blood cells. The lymphocyte and monocyte sub-populations (CD4, CD8, CD20, CD14, CD16) look as expected, but although we are getting a very nice yield of granulocytes (determined by FSC/SSC), they're almost all CD66b+ and CD16-, which leads me to believe they've become activated and are beginning to go through apoptosis. As we're running healthy young volunteer samples I think this high level of activation may be a the result of the protocol itself. Has anyone else experienced this?
Should I use two separate protocols, one for PBMCs and a gentler (and faster) one for the PMNs?
In addition, has anyone had any success with freezing granulocytes and still being able to detect markers by flow cytometry?
Protocol Details:
All cells undergo erythrocyte lysis in a commercial buffer and fixation in 4% PFA before staining. The wash buffer contains 2% BSA and is blocked in 10% human serum.
Dear Meaghan,
granulocytes don't usually survive freezing. I think the best way to go about it is to stain in full blood, and then lyse RBC after antibody incubation, wash and then FACS. This works very well for us
Hi Meaghan Roy-O'Reilly ,
Please see our article it could be useful to a part of what you asked!!!
https://www.researchgate.net/publication/266560460_The_effect_of_sample_storage_on_the_Peroxidation_of_Leukocytes_Index_Ratio_%28PLIR%29_measure
Article The effect of sample storage on the Peroxidation of Leukocyt...
Dear Meaghan,
granulocytes don't usually survive freezing. I think the best way to go about it is to stain in full blood, and then lyse RBC after antibody incubation, wash and then FACS. This works very well for us
Dear Meaghan
May be you are using a CD16 antibody with weak or no staining of neutrophils. Suggestion to use a monoclonal like 3G8. This should give a strong signal on granulocytes. See https://www.researchgate.net/publication/45367697_Standardized_single-platform_assay_for_human_monocyte_subpopulations_Lower_CD14CD16_monocytes_in_females
for details.
Yours
Loems
Article Standardized single-platform assay for human monocyte subpop...
Dear Meaghan,
Maybe a silly suggestion: did you try staining before PFA fixation? I noticed that NK cells are CD16+ using your protocol, but maybe granulocytes have a different epitope sensitivity to PFA?
Hi Albert,
We're not using an FC receptor blocker in this procotol.
I would highly recommend staining before fixation. How long is your RBC-lysis incubation?
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