I need to isolate and purify neutrophils from peripheral blood of nonhuman primates (cynomolgus macaque). The only published protocol I've come across is from the 2014 Neutrophil Methods and Protocols (link below) that recommends CD66abce microbeads from Miltenyi. Considering how easy it is to activate PMN, I try to avoid positive selection whenever possible. If anyone has suggestions or leads on a superior isolation method (e.g. density centrifugation, negative selection) that works for NHP, I'd really appreciate the help.
Thanks.
https://www.ncbi.nlm.nih.gov/pubmed/24504944
Dear Pat,
I have never isolated neutrophils from non-human primates, but typically used Percol gradients (which can be adapted in case the cell density is different for non-human primates), and negative magnetic selection using MACS columns and custom antibody cocktails (DOI: https://doi.org/10.4049/jimmunol.168.9.4701).
Using this method, we never found any neutrophil activation in our samples.
One important change to the published protocol that you cited - you should never (!) "Flush out the magnetically labeled cells by depressing the plunger into the column.", as this WILL activate your cells. Instead, let gravity do its work.
Hope this helps.
Best,
Svenja
Thanks, Svenja! I probably should have mentioned that I've isolated PMN from human and mouse using both density centrifugation and magnetic separation many times. I cringed at the same line in that protocol!
I suspect that you're right about using Percoll and I'll probably need to adjust the % based on different PMN densities for each species. I appreciate the feedback!
Hi Pat, you're very welcome.
Yes - I thought you might have had isolated neutrophils before... Happy that you share my thought on that line...
I have adjusted a percol gradient before to separate neutrophils based on their maturity. There was a great paper, where they had some more info on the different densities, but I cannot seem to be able to find the reference right now...
Instead, I found this:
J Pharmacol Toxicol Methods. 1995 Apr;33(2):91-100.
Separation of neutrophils from blood in human and laboratory animals and comparison of the chemotaxis.
Sugawara T1, Miyamoto M, Takayama S, Kato M.
They isolated neutrophils from different species (including monkeys), and they have a pretty detailed list of the different gradient fractions, where the different cell types accumulated.
Should be quite helpful, if you do go for adjusting your gradient.
Best of luck.
Svenja
Svenja Dannewitz Prosseda was this in humans? If so, can you share the modified protocol(s) you use?
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