Abstract
This is a general protocol to stain whole human blood for flow analysis with minimal spontaneous activation of monocytes. This protocol was developed or modified in Dr. Anne Davidson’s lab at Feinstein Institute for Medical Research.
Keywords: Human, Flow cytometry, Monocytes, Whole blood
Materials and Reagents
Equipment
Software
Procedure
Gating strategy
Acknowledgments
This protocol was developed or modified in Dr. Anne Davidson’s lab at Feinstein Institute for Medical Research, NY, USA. This work was supported by grants from the NY SLE Foundation (RB), Rheuminations, NIH AI082037 and AR 049938-01, NIH (PO1 AI51392 and the Flow Cytometry and Protein Expression and Tetramer Cores of PO1 AI51392).
References
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Yes. It has become a very exciting field of research in immunology research.
You are right. Thank you for pointing it out.
Dear Hanan,Thank you for pointing it out. With Dr. Zheng Liu's permission, "lymphocytes" has been replaced with "monocytes" in the protocol.
Please try to stain before lysis. The double negative cells could be contamination from other population. We try to gate them out using a dump gate containing T, B, NK cell markers, but you always get a few of those cells regardless.
Sorry I have no experience in culturing monocytes.
Yes. You can still find the three populations after ficoll. Whole blood staining has two advantages I can think of. 1. it is easy and saves your time.2. it doesn't activate monocytes. In my hands, Ficoll activates monocytes which upregulate their surface expression of active form of CD11b and maybe other molecules. So it may not suits certain experiments. Dr. Z. Liu
I am glad that you thought it was interesting. As to you questions, 1. whole blood staining does not work with all antibodies or cell subsets. For instance, we tried and failed to stain human DC subsets with whole blood staining using a 9 antibody mix. So pilot experiments are needed to determine if your antibodies are compatible with this method.2. I have no experience with anti-CD44 antibodies.Hope my answers are helpful.Regards,Dr. Z. Liu
Thank you
No, I have no experience with CD11b and Ly6C in mouse whole blood. As to RBC lysis, I recommend BD’s PharmLysis which usually results in a pretty complete lysis. But I only used it before staining the cells with antibodies.
As far as I know, CD86 is an activation marker which is not limited to monocytes. Therefore I personally don’t think you can get all monocytes using CD86.
I personally have no experience with CD66. However, CD66 is expressed by many cell types including monocytes, neutrophils, and epithelial cells. So I am not sure how specific it would be as a monocyte marker. In addition, CD14 and CD16 allow us to distinguish the three subsets of monocytes which are phenotypically and functionally different from each other. I hope this helps. Thank you for your question.Zheng Liu
Thanks for pointing this out! I have corrected it in the protocol text.