I do not have experience with this, however it seems that growing the cells on a coverslip and staining with a-DNA antibody or other DNA binding reagent might work

http://www.abcam.com/ds-dna-antibody-hyb331-01-ab27156.html

One of the methods we use in our lab to detect Neutrophil extracellular traps is western blot using anti-H3 antibodies, which worked very well. There are other ways to detect  it., eg. immunofluorescence.

Hey,

thank you very much for your answers. I am not sure if I could quantify NETs using these methods. However, I am using ELISA for dsDNA and NE and it should be specific enough.

Anyway, thank you once again. I appreciate your help :)

Check this artice: https://www.ncbi.nlm.nih.gov/pubmed/?term=kraaij+method+neutrophil

Detection depends on what types of samples you are using.  In my experience working with NETs, neutrophil elastase alone is not sufficient to determine NET release because its release can be independent of NETosis.  If you are looking in serum, I would suggest using a cit-H3 antibody to detect citrullinated Histone H3 since at this time; it is considered a hallmark identifier of NETs.  Since you are using neutrophil elastase as a marker, perhaps you can run a sandwich ELISA using anti-citrullinated histone H3 and anti-neutrophil elastase antibodies, you could also use myeloperoxidase in place of NE.  There are a few ways to go about achieving your aim, but without knowing any details of what you are working with, I cannot give further suggestions.

Thank you very much for your answers,

I am working with induced NETs by PMA usually. I was thinking about ELISA detecting both dsDNA and citH3. I believe that could be the best option (plus additional measuring of  the DNA concetration). What do you think? Is it a good idea?

Thank you

If you are isolating neutrophils and inducing NETosis with PMA, that should be okay.  If you are performing an analysis using a disease model, it may not accurately reflect physiological levels of NETs.  NETs have been implicated in endothelial and epithelial damage and other aspects of histotoxicity.  Extraneous DNA trapped within NETs could result from collateral tissue damage and not from actual neutrophils that underwent NETosis.  Keep in mind, eosinophils, macrophage and mast cells also release extracellular traps and it has been suggested that basophils release traps as well.  Because of this, it is common to use MPO or NE in conjunction with cit-H3 to detect NET release.  Macrophage can also release NE and MPO, but hypercitrullination of histone H3 at residues 2, 8 , 17 and 26 by PAD4 make this marker unique to neutrophils. 

Long story short, yes anti-dsDNA/cit-H3 antibody ELISA will be sufficient if using isolated neutrophils.  If you plan to test serum, plasma, BAL fluid, etc. for NETs, I would use MPO or NE in place of dsDNA detection.  I hope this information helps you. Let me know if there any other suggestions I can offer.  I also have experience imaging NETs using immunofluorescence on frozen tissues.

Thank you very much for the valuable information. I am isolating PMNs from diabetic patients and inducing NETs with PMA or serum (20% probably?). There are some critical steps, including multiple washing and using nuclease. Despite these steps, I hope the quantification will be meaningful and right.

I am planning to image NETs. Unfortunately, do not have a lot of experience with fluorescence microscopy so at this point I am just “playing” and trying to figure it out. Anyway, here is my protocol. Do you think this could work?  First, I label PMN with PKH26 red fluorescent cell linker and then I stimulate the cells. After 3-4 hours, I fix, block (5% goat serum) and label them with antiNE (1:200 diluted)  and anticitH3 (1:300 diluted) antibodies. As a secondary antibody I use goat antirabbit Ab conjugated with A488 (diluted 1:500). And as a final step, I add Hoechst 33258 (1ug/ml), wash and image the cells on ScanR. Unfortunately, it is not good yet. I guess I have to use more concentrated primary Abs and probably less Hoechst and PKH26, stimulate shorter, use less cells. As I was saying, I am still at the beginning and a lot of optimization is ahead of me. I would be nice to use microscopy for quantification, but I am not there yet. Till then I have to rely on ELISA. :)

Once again, thank you very much for your help. I really appreciate it.

Your staining method seems good, not sure what Ab you are using for cit-H3 detection, I used Abcam (ab5103) for my investigations and if I remember correctly I used a 1:333 dilution so it sounds as if your primary for cit-H3 is good.  I used DAPI as opposed to Hoechst (not for any reason other than I had a lot of DAPI already in the lab), the stock concentration was 1ug/mL and I used a 0.05% concentration in my staining solution.  Just remember to take great care during the washes, NETs are extremely fragile and can be lost when washing.  There is a wash method Brinkman et al. 2010 published in the Journal of Visualized Experiments that I really enjoyed.  Washes were performed by transferring the cover slips with fixed NETs onto drops of PBS on Parafilm sheets covering a test tube stand.  I recommend giving this method a try while you continue to optimize your staining protocol.  As far as number of cells, I could suggest starting around 2 x 10^5 cells/500uL and titrate from there. 

It sounds as if you are getting close, keep it up the hard work and best of luck on your  ELISA and IF staining.  :)  If you don’t mind, let me know what worked for you, it's always great to hear new methods of detection.

Until now I was doing the IF staining in glass bottom Petri dishes or multiwell plates. But I guess, I can try do it like in the video from Brinkman.

I was planning to use commercially available ELISA for MPO and citH3. Unfortunately, they decided to discontinue selling the kit because of inconsistent results. So I will keep looking for the right ELISA.

I will let you know as soon as I achieve some success in the NET quantification. 

Thank you very very much, you have been very helpful :)

Glad I could help, best of luck!

Hey Maria,

would you be so kind and give me a contact for that person?

Thank you very much :)

Hello,

I am doing IF on frozen mouse brain sections to view NETs. I am using ab5103 at 1:100 dilution. But I see absolutely nothing. The infection works in the brain, because we see the parasite. So I dont know if the Ab that I use is too diluted, or did somebody do ethanol fixation for the Ab to reach the site?

Thank you very much :-)

Hi,

anti-citH3 (1:200) works ok in my in vitro induced NETs but I have a weak signal from anti-NE (1:200)  antibody. Are you able to see at last DNA? Unfortunately, I have no experience with ethanol fixation so I can not tell if that is the reason. However, I know that NETs are detected in other tissues, such as pancreas, so maybe you could check these publications to see what methods they are using?

I am sorry I can not be more helpful

Good luck with the detection :) 

Hello,

I measured NE activity (Cayman), NE concertation (Abcam), LL37 concertation (Hycult Biotech), histone-associated DNA (Sigma) but I am still not sure if this is the way how to quantify them. I even tried Image Stream analysis and none of these approaches is good enough I am afraid. A combination of these methods could probably give complex information about how your NETs look like .But for the quantification, the microscopy is probably the best way.

I hope this helped and good luck with your experiments J

Two questions to Zuzana Parackova or anyone using the CAYMAN NETosis kit...

It works by allowing cells to form NET, washing intact cells to remove soluble proteins from media, adding S7 nuclease to release Elastase into the supernatant, which is quantified by a colorometric substrate, and the Elastase activity is proportional to NET formation.

1. What about the DNA that is also released. Do you think there is enough dsDNA that one could assay THE SAME supernatant with a fluorescent dye like Pico-Green and quantify the amount of DNA present? It should be proportional to the NET formation and give and "verify" the Elastase result.

2. S7 nuclease appears to target both ssDNA and dsDNA, but is more active on ssDNA (https://www.thermofisher.com/order/catalog/product/EN0181). So if one lets the reaction run to completion there should be only free nucleotides, but if the reaction is limited then there should be dsDNA present. Does this seem possible? If one chops enough NET to liberate Elastase while preserving enough dsDNA, then one might be able to measure both Elastase activity and dsDNA in the same sample. Is the kit robust enough to accomplish that?

These questions get at the goal of having a dual read-out from the same sample. One might need that if changing cell types or drugs or anything else that might attenuate the Elastase read-out, in which case one still has the DNA readout....

Hello Luis!

Thank you for the questions! I will try to respond as well as I can.

At first, I tried to measure DNA using nanodrop and it was imposible. I had no signal. I have not tried dyes like Picogreen, so I am not sure how this would work. I am using for other experiments NETs cleaved by other enzymes -AluI, NdeI, PacI and I am able to quantify DNA using nanodrop.

I do not think that it should be proportional, the amount of NE and DNA. I used another ELISA kits form other companies, detecting histone-associated DNA, NE and LL37. For example, diabetic patients had more histone-associated DNA, but low levels of LL37 and lower NE activity compared to controls.

Cayman kit measures NE activity, I would suggest to measure also other proteins, especially their concentration, as well as histone-associated DNA.

I was able to detect histone-associated DNA in the samples. So it seems that in 15 minutes of cleavege, you still have enough dsDNA. It would be helpful to run electrophoresis to detect DNA fragmentsts to be sure. I performed electrophoresis only from AluI digested NETs, but there are clear bands of DNA on the gel. However, I think you should be able to detect dsDNA as well as NE activity in your samples.

I hope the answers are helpful. If not enough, please do not hesitate to ask

Zuzka

There are many aspect to check the NETs release in real time and in fixed cells and tissues.

If you have specific question, I can explain you and provide detail protocols.