Recently I performed RBC lysis to obtain WBCs from human whole blood. For my application, I use immunofluorescence to identify neutrophils and lymphocytes from this WBC population. The antibodies I use are CD3 (T cells) and CD19 (B cells) for lymphocytes and CD16 for neutrophils. I observed non-specific antibody binding, for example, lymphocytes were staining for CD16. Also the expression levels of all the antibodies were quite low. When I use density-gradient media such as Polymorphprep for separations, I don't face these issues. I am wondering whether there is evidence to say that lysis might be the cause here and if so, I would love to know why.
Any insights here would be greatly appreciated.
~Ani
Dear Anirudh Gangadhar ,
In my experience, it is not very likely that RBC lysis causes such considerable changes in the staining levels of CD3/CD19.
One thing I could note here is that CD16 is an FC receptor, and some antibody clones may have strong affinities to it. So, you could probably try an FCR blocking using for example one of Miltenyi products.
https://www.miltenyibiotec.com/US-en/products/macs-sample-preparation/fcr-blocking-reagents.html#gref
Then, if you are unsure of the choice of another neutrophil marker, you could look for one in the following link (I personally prefer CD66b).
https://www.bd.com/documents/bd-legacy/catalogue/biosciences/DS_Human-Mouse-CD-Maker-Biosciences_CT_DE.pdf
If you think your observations are dependent on your experimental setting, then you could probably think of troubleshooting by yourself. For example, you could start from preparing RBC-lysed cells and density-separated PBMCs at the same time, from the sample blood sample, for flow cytometric analysis without staining the cells with any antibody.
When you said that CD16 is staining lymphocytes, what do you mean exactly? That you see CD16 in the lymphocyte gate on SSC/FSC? Or are they staining cells positive for CD19 and CD3? If it's the latter, then you do have an issue. If it's the former...it could just be a gating issue.
Tetsuo Tsukamoto I considered CD66b while making my selection but as it is a granulocyte marker, it would stain for eosinophils too. I needed something that would only discriminate neutrophils and not pick up other granulocytes and therefore went for CD16.
Shen-An Hwang I should have mentioned that the method I use is immunofluorescence (IF) and not flow cytometry (FC). Yes, its the latter.
So, I suppose you intended to discriminate between neutrophils and eosinophils by staining with anti-CD16, because they have similar side scatter light signal levels. Then, I could suggest testing another anti-CD16 conjugated to a different fluorophore to troubleshoot the observed staining of lymphocytes with anti-CD16.
Please also consider answering the question by Shen-An Hwang .
In humans, CD16 is present on peripheral blood natural killer cells which are lymphocytes and will appear in the Lymph gate. They are CD16+, CD56+. The marker is also present on some monocytes (usually CD14) and may indicate activated monocytes
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