I am trying to stimulate neutrophils with PMA using the Abcam NETosis kit (ab235979). I treated my neutrophils with a range from 20 nM to 100 nM PMA for 4 hours at several cell concentrations. I see no effect at any PMA concentration, the elastase concentration is the same as untreated cells. I was thankfully able to see a difference in elastase activity that correlated VERY NICELY with cell number. So at least I know the assay is working. I just do not know why the PMA in the Abcam kit is not stimulating NET formation?
I was not sure if it could be the media - I used RPMI 1640 Medium, GlutaMAX Supplement, HEPE (Thermo Fisher). I read a paper saying glucose is important for NETs formation, but RPMI should have glucose so not sure what the problem is. Any suggestions would be helpful.
I have also heard serum inhibits NET formation, but you can get heat-inactivated which would be ok to use. I used the BSA that was included with the Abcam kit because when I asked previouly, they confirmed it was heat inactivated. So, this should not be the problem although I could try not using the serum.
Any help would be much appreciated.
Hej, we have seen that heat-inactivated FBS inhibits ROS production in neutrophils. So it could be that the FBS interferes with your assay?
https://www.frontiersin.org/articles/10.3389/fmicb.2020.00065/full
Dear Megan!
Please You look at this article:
Article In vitro induction of NETosis: Comprehensive live imaging co...
Table 1
Overview of in-literature described in vitro NETs inducers.
Try using a higher concentration of PMA and a different time
120–1620 nM (74–1000 ng/ml) during 10 min-4h Yes[147–166]
100000 nM (6168 ng/ml) during 10 min-24h Yes[14, 167, 168]
or anather inducer, for example, H2O2
10000 μM during 4-5h Yes [54]
0.03% during 3h Yes
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