I isolated neutrophil from healthy blood and all of my neutrophils are positive for CD16, CD62L and CD11b antibody but only 60% of neutrophil are positive for CD11c, 71% for CD54 and only 10% for CD88 in un-stimulated condition. I am keen to know that at what extent these surface markers are usually present on the surface? And does all neutrophil express CD88?
Regards
Ravi
I've been measuring CD88 on the surface of neutrophils for many years -in unstimulated, healthy neutrophils it is usual to find 100% of neutrophils positive for CD88 -indeed even when you activate them they usually remain positive but with reduced expression.
Activation -by exposure to LPS (e.g. contaminated reagents for preparation) will reduce CD88 expression. A prep that activates neutrophils (e.g. not handling them gently) will also reduce CD88 expression.
One simple way to look at this would be to do a whole blood stain -the neutrophils should be largely unactivated, and you can compare your CD88 expression (and indeed CD62L/CD11b) between your prepped neutrophils and those in whole blood to see if it is your prep that is the problem.
Let me know if you want any protocols
I would like to suggest you to "gate" your neutrophils first with CD15 and then measure the frequency of your marker within CD15 positive population. The result varies a lot between morphology gated and CD15 gated neutrophils.
Thanks Everyone for answers, I actually gated CD16 positive population and then I was looking at the expression of CD88 and other markers. In that I was only getting 10% positive population for CD88.
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