Hello,
I am new to using FACS for sorting and did some browsing through Google, but I could not find a proper protocol for separating white blood cells into the following populations:
- Neutrophils
- Eosinophils
- Basophils
- Monocytes
- Lymphocytes
Could someone suggest a protocol as well as antibodies to use for cell sorting? Could we start by sorting monocytes / granulocytes / lymphocytes using light scatter, and then separate the granulocytes into neutrophils, basophils, and eosinophils using antibodies?
You may use antibodies that will help you to identify these population together with scatter. First you use CD45 as a pan leucocyte marker, so that you make sure there are no red blood cells, plasma cells, platelets in your sorted cells. The markers we use are: Neutrophils-CD15, CD24. Monocytes-CD14, Lymphocytes-CD3 for T cells, CD19 for B cells, basophils - CD63. Eosinophils can be discriminated by CD45/SS, or FS/SS, we have not used yet, but CD193 maybe a good marker for eosinophils. I assume that, you want to sort human cells. Basophil identification maybe a bit tricky, you need to experiment. For basics of immunophenotyping I suggest that you read the book that was written by Marion Macey. "Flow Cytometry: Principles and Applications".
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