Hi Miron,

if you only need to quantify NETosis (and not look at cell markers by flow cytometry) you could allow the neutrophils to adhere to poly-l-lysine coated plates before inducing NETosis by PMA (we usually do 20 nM for 3-4 hours). This will enable you to easily wash away any added inhibitor and/or PMA. Released NETs can be quantified either by IF or by fluorimetry (after partial micrococcal nuclease digestion to release the NETs from the plate). You could read more about those methods in one of our recent papers (Lood C et al 2016, Nat Med) were we analyzed NET-induction in presence of different inhibitors using several stimuli.

Please let me know if you would need some further details, and best of luck with your experiments.

Regards,

Christian Lood

Dear Christian,

Your reply escaped our radar and we would like to go back to the experiments with the Neutrophils.

We saw that you were using poly-L-lysine coated tissue culture plates or coverslips.

However, when we tried to seed the cells on Poly-Prep Slides (poly L-Lysine coated glass slides P0425-72EA Sigma) the cells didn't adhere well or that the cells were washed away or completely dried up.

We will appreciate if you can please send us a detailed protocol (with the coating). We were told that maybe we have to add Laminin to the slides?!

Thank you very much,

Miron & Ariel

Dear Miron,

I have unfortunately mainly been using tissue culture plates (also for microscopy), but hopefully you could still benefit from the information. Otherwise, let me know and I'll reach out to some of our collaborators doing confocal microscopy of neutrophils/NETs on coverslips.

I have pre-incubated tissue culture plates (48 well flat bottom #25-108 from Olympus plastics) with 100 uL poly-L-lysine (Sigma #P4707) for 1-2 hours at 37oC or at 4oC o/n. You then wash away non-bound poly-L-lysine with PBS three times and allow the plate to dry.

If you then add your neutrophils (200 uL, 1x10^6/mL) they will adhere firmly within 30 minutes at 37oC and you'll not be able to wash them away.

I know of collaborators not using poly-L-lysine as well, but in our hands the neutrophil adhesion and NET inducing ability has been more consistent with poly-L-lysine.

Please let me know if you have any further questions on the protocol.

Best regards,

Christian

Dear Christian,

Thank you very much for your prompt answer.

We will try again to work with the Poly-Lysine slides (Sigma) according to your guideline (1X10^6 cells/ml).

I would like to know too how you can visualize NET in the tissue-culture plate (isn't it too thick for the confocal microscope)?

All the best,

Miron

Hi Miron,

correct, you can't use those conditions for confocal microscopy. We have mainly been doing IF microscopy (our current microscope can do x40 also for tissue culture plates), and it has been convenient to do 48 well tissue culture plates for quantification (MNase digestion followed by DNA and MPO-DNA quantification) to avoid tedious (and subjective) quantification by microscopy. It is nice with confocal but in my hands x40 IF microscopy has been more than sufficient to visualize NETs.

If you need to do confocal microscopy we (and others) have been using poly-l-lysine covered/coated glass slides and/or culture slides instead of the tissue culture plates. I think there should be plenty of options on the market, but we have among others been using Falcon Culture Slides (4 chambers, #354104). Or you could simply coat an ordinary glass slide with poly-L-lysine, which according to the literature should work fine. Unfortunately I do not have as much experience with those slides, but in my hands the neutrophils seem to adhere very firmly also to those slides, perhaps even better than to the tissue culture plates.

In all, if you don't need to do confocal I would give the tissue culture plates a try, otherwise I suppose you'll have to give it another try with the glass slides. Note that you'll have to do extensive washing to get rid of non-bound poly-L-lysine as well as allow the glass slide to dry before adding the cells.

Sorry I could not be of any more help for the confocal part,

Christian

Dear Christian and Miron,

I recently did some pilot NETosis runs. During these runs, I noticed that the PMA stimulated purified neutrophils (which generate beautiful NETs) adhere more firmly to the 8 well chamber microscopy slides while the cells that remain unstimulated or stimulated under different test conditions are lost quite a bit during wash processes.

I have been coating Ibidi 8 well chamber slides with 400ul of poly L-lysine at a concentration of 15ug/ml (recommended by company) followed by 3 washes with 1X PBS. The poly L lysine stock is same as what you mentioned Christian, P4707-50ML.

Christian Lood : In my next run, I'll add 100ul of stock directly onto slides like you mentioned. I am wondering what do you mean by extensive washing. Is it like 3-5 washes ?

Miron Krimsky : Any updates on what worked best for you?

Thanks to all,

Anita

Dear Anita,

It is important to remove all poly-L-lysine that has not bound to the plate due to potential cytotoxic effects in solution. We usually allow for binding of poly-L-lysine o/n at 4oC or 37oC for 1 hour. Once bound we remove excess poly-L-lysine, wash once with PBS and allow for the plates/wells to dry completely prior to adding our cells. I hope this clarified your question, and I'm sorry for the delayed reply.

Best regards,

Christian