Hi Ang Lin,

It's a challenge to phenotype cells from less-studied species due to lack of literature and reagents. so I hope you will succeed.

To my knowledge based of human cell isolation, ficoll is not selective for isolating PMN s you will get all PBMCs as well. So you my have to add a layer of histopaque or percoll to make a step wise gradient or at least get a better enrichment for PMN.

Did you check your cells microscopically? That may help you to evaluate how well you isolation works.

In humans you typically won't expect high levels of CD14 on PMN, but who knows macaques may be different.

So, in your case I would look into checking and/or improving mycell prep first.

Thank you Gerco! I agree with you, it is challenging to phenotype rhesus subsets sometimes. 

The reason I looked at neutrophils in PBMCs fraction is that we are doing some research on the low density neutrophils. So far I only measured CD14 on low-density PMN, haven't done this on normal density PMN. Thanks for your advice! I think it is interesting to measure CD14 on normal density PMN also, may be it will show some difference. 

it would be good to include a secondary marker that discriminates between pmn and eg monocytes in addition to cd14 and ssc. 

An Ab that recognizes both human and mouse probably binds to a conserved region and will thus have a good chance to work for macaques too. In general anti human abs may work for you as the identity between human and macaques is high? 

Hi Joana Dias

we now think rhesus neutrophils have a dim expression of CD14, at a much lower level than rhesus monocytes.

Some other studies also showed this: Lafont BA, et al. Cytometry. 2000; Elbim C, et al. Retrovirology. 2009.

Hope this would help.

Ang