I'm doing experiments on neutrophils producing NETs. In the preliminary experiment, I successfully induced NETs from rat neutrophils using PMA. When I used ADP preactivated platelets to stimulate neutrophils, NETs were produced much less than in the PMA group. And the cells would cluster together after the stimulation, which I think might affect the fluorescence intensity.
I wondered whether platelets and neutrophils needed to come from the same rat, and if so, whether platelets in the resting blood would be altered during neutrophils isolation?
By the way, Neutrophils are taken from bone marrow and I used Sytox Green to indicate the formation of NETs.
Your advice and experience are greatly appreciated!
Sorry!
How did you activate platelets?
You can also use the released products from platelets. Yours.
Mourad Aribi
Dear Professor Aribi,
Thanks for your advice!
I isolated platelets from the whole blood using a two-step centrifugation, then adjusted the platelet count to 3×10*8/mL with a Tyrode solution. After standing at room temperature for 30min, ADP (final concentration: 10μM) was added and incubated at 37℃ for 10min. ADP only and resting platelets were set as control groups.
But I have another question. Platelets are very easy to activate. Don't resting platelets spontaneously activate when neutrophils are added? But a lot of the literature uses resting platelets as a control, and I'm not sure that's feasible.
Best wishes!
It'd be better if the platelets are extracted from a different animal. As far as agglutination (clumping) is concerned it is quite common after PMN activation. Try adding BSA to the incubation medium.
Vipul Batra
Thank you! I used RPMI 1640 medium containing 10% fetal bovine serum. Does the agglutination of neutrophils make a big difference to the results?
Fang Yafei Yes, in a way it would. The NETs wont be discernible clearly which would make it very difficult for you to assess the effects. PMNs get activated pretty easily and start dying soon after isolation which usually results in clumping. Also please check out this article : DOI: 10.1186/s12964-018-0235-0.