Looking to compare several methods -- any advice appreciated!
First, you'll want to get the red cells out of the way with a lysis buffer. Then, optimize yourself a scatter gate that isolates the granulocytes. At that point they will be very "pure" and you can ask whatever question you have in mind about them with different colored antibodies. I can't really advise too much about which markers to look at, without understanding more about your research questions.
FACS markers to be used include: CD16+Lactoferrin+ or CD15+Lactoferrin+ (LF has to be stained intracellular). LF is unique to mature blood Neutrophils.
As John said you simply have to get rid of red blood cells by a lysis buffer, for example ammonium chloride. you can then use several antibodies to test your granulocytes, also depending on which granulocyte you want to study (neutrophils, eosinophils or basofils). Finally, since granulocytes are very delicate, you should include a viability probe such as 7AAD.
It depends on which markers you are going to detect. If you want to obtain granulocytes in their most natural state, leave your heparinized whole blood sample to gravity at room temperature after you lay it over a density gradient solution (Ficoll), after exactly 40 minutes, collect the neutrophile rich upper 600 microliter. Count the number of cells. Then stain your cells. At cytometry analysis, you can gate on neutrophils on FS/SS or use SS/CD15 gate.
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