The best way to gate out granulocytes is with CD66b (clone G10F5) although some groups are using the less specific CD66 (clone CD66alpha-B1.1)
When you thaw out blood samples banked with Smart Tubes or Smart Tube buffer systems and follow the standard protocols you are left with are total leukocytes which are PBMC + granulocytes. If your stain includes CD66b (specific to granulocytes) and you gate out the CD66b positive cells then what you have left are essentially the PBMC you would recover from a ficoll prep (ie. mostly lymphocytes and monocytes). Make sure you use Fc block first (Biolegend's FcX reagent has been shown to work very well).
When conducting pilot experiments with Smart Tubes or Smart Tube buffer systems it is important to include a stain like CD66b as a way to positively identify the granulocyte compartment as FSC and SSC is not sufficient to gate them out in samples banked with Smart Tube systems. Degranulated granulocytes are particularly problematic and can have FSC and SSC that are indistinguishable from the lymphocytes and monocytes.
Note that some of these groups have found significant clinical value analyzing the granulocyte compartment:
Smets, Tina, et al. "Deep Profiling of the Immune System of Multiple Myeloma Patients Using Cytometry by Time-of-Flight (CyTOF)." Multiple Myeloma. Humana Press, New York, NY, 2018. 47-54.
Behbehani, Gregory K. "Cell Cycle Analysis by Mass Cytometry." Cellular Quiescence. Humana Press, New York, NY, 2018. 105-124.
See these and other references on our Publications webpage: https://www.
Keywords: granulocytes, gating, resolving populations, degranulation, problems gating lymphocytes and monocytes, low recovery, where are the lymphocytes, where are the monocytes
0 Comments