I tried 100nM of PMA on 200 thousand neutrophils and incubated 30 minutes (15 minute in incubator and 15 minute after adding PI at room temperature) and all my cells died after that. I am looking for some other alternative to PMA.
Hi Ravi,
PMA is a well-known inducer of NETosis, a specialized form of neutrophil necrosis. We usually do not see NETosis as early as after 30 min, but tend to use lower concentrations of PMA (20 nM) for our studies..
With regard to other agonists, it depends on what you would like to study, and if working with mice or human neutrophils. fMLP is commonly used to induce neutrophil activation and study Ca2+ signaling and ROS generation.
However, I would probably recommend a standard TLR agonist. If working with human neutrophils, PAM3CSK4 (TLR1/2), R848 (TLR7/8) and LPS (TLR4) will induce robust neutrophil activation, and upregulation of granular proteins on the cell surface. They can all be used at about 1-2 ug/mL, but you may have to optimize it in your lab.
Hope that could be of some help.
Best regards,
Christian Lood
Thanks Christian, I also want to share with you that I am usually taking 3 hours in isolation by percol method and 1 hour in antibody staining so total 4 hours. Do you think that this can also be a reason as before adding PMA they may already in active state. I also tried 20nM PMA still 96% cell died. I want to know how to cool them I mean after isolation what should I do that they remain calm and not get activated. Also what best stimulating agent you suggest me by keeping my isolation protocol in mind.
Hi Ravi,
neutrophils are very sensitive and do undergo apoptosis fairly quickly. But I'm still surprised by the rate of cell death you are experiencing. Would you be willing to test another isolation protocol? The protocol we are using (Polymorphprep) should give you very pure human neutrophils within 1-1.5 hours. Would be happy to send you our protocol (or you could find it in our papers). And depending on what antibodies you are adding, but 30 minutes on ice should be more than sufficient for antibody labeling of cell surface proteins. I think the key points would be to reduce the isolation procedure in particular and keep the neutrophils on ice as much as possible to reduce unnecessary activation. You may also want to look into your lysis protocol (I would assume you lyse contaminating red blood cells?) as prolonged lysis will also cause neutrophil death. Would be happy to share our protocol - maybe easier so through email ([email protected]).
If your neutrophils are already primed/activated upon isolation it will be more difficult to see differences upon addition of the stimuli. But all the stimuli I recommended are strong agonists and will induce a robust degranulation and up-regulation of several cell surface markers (we usually analyze CD66b and CD11b expression). In our hands, for human neutrophils, 1-2 ug/mL of R848 for 3 hours is a very strong activating stimuli. But you could as well do PAM or LPS, depending on what stimuli you have at your disposal.
Best of luck,
Christian
Hello this chain has been helpful. I have an additional question regarding NETosis and stimulation with PMA.
My question is regarding the NETosis Assay kit from Abcam (ab235979) measuring neutrophil released elastase. I have run this experiment twice now. The first time I saw PMA stimulated the neutrophils 40-50% above unstimulated control and this was satisfactory for my downstream treatments and analysis.
The second time I ran this experiment I only saw 10% stimulation with PMA. I was wondering your thoughts on this. The standard protocol, I believe if my calculations are correct, uses a final concentration of PMA of 50 nM/mL. My calculations are based on diluting 1uL of 1 mM PMA into 5mL PBS equaling 500 nM (and after the 100 uL dilution into 900 uL of sample) this would come out to 50 nM PMA per well.
Should I try higher PMA concentrations? Or would you suggest less dense plating of neutrophils. In the protocol it says 1x10^6 neutrophils per mL and plating 900 uL. I see in the literature people use 200K to 500K cells per well of a 24-well plate this contrasts with the suggested 900k cells per well in the Abcam protocol.
Cells and kits are expensive so any help on your suggested change, higher PMA, less confluent neutrophils, etc. would be greatly appreciated.
Ravi Lokwani This thread is already a few years old and I hope you solved the issue (if yes, how?). However I wonder if the neutrophils may have died because of the PI - adding it to cells for 15 min would probably kill most cell types, wouldn´t it?
- Badges
- Science topic
- Similar topics
- Computer Science and Engineering
- Bioinformatics
- Bioinformatic Tools