I use RosetteSep™ Human Monocyte Enrichment Cocktail without CD16 depletion to isolate the monocyte cells from normal peripheral blood mononuclear cells. Initially I used CD45, CD3, CD19 and CD56 to check the isolated cells. More than 95% of the isolated cells are CD45+CD3-CD19-CD56-. Then I used CD68, CD163 and CD14 to detect the percentage of monocytes in the isolated cells. But I find only 60% of isolated cells are stained positive for CD68, CD163 or CD14. So what are the remaining cells (CD68-CD163-CD14 cell population) and How do I test the purity of monocytes in the isolated cells? Thanks.
I would say that CD14 should normally be good enough to identify monocytes in PBMCs. Can you show what the FSC/SSC and CD14 staining look like? Maybe you have granulocyte contamination. Or alternatively the CD14 staining is not strong enough? I would always perform the same staining on the pre-isolation sample so that you are sure that your staining works.
CD-marker expression is certainly important (most people would probably argue that CD14 is enough), but to investigate what the possible contaminants are, I would probably use FSC/SSC plots. In addition, have you looked at granulocytes (the largest cell fraction in peripheral blood)? I also assume that you included isotype-controls etc. to make sure that CD3- is really CD3- etc.?
Lastly, your results (60%) are not far away from the "typical" result you are supposed to get with this method (70-80%). Magnetic cell sorting (selection or depletion techniques) might give populations with higher purity, especially using 2-step procedures (purifying the sorted cells a second time).
Hope, this helps a little.
As mentioned above, SSC/FSC should help you to see if the contaminants cells are monocytes; to check for neutrophils I use CD66b/CD16 (N are double positive), monocytes are negative for CD66b.
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