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 JoVE Immunology and Infection

Neutrophil Extracellular Traps: How to Generate and Visualize Them

1, 1, 1,2, 1,2, 2

1Core Facility Microscopy, Max Planck Institute for Infection Biology, 2Cellular Microbiology, Max Planck Institute for Infection Biology

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    Summary

    Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

    Date Published: 2/24/2010, Issue 36; doi: 10.3791/1724

    Cite this Article

    Brinkmann, V., Laube, B., Abu Abed, U., Goosmann, C., Zychlinsky, A. Neutrophil Extracellular Traps: How to Generate and Visualize Them. J. Vis. Exp. (36), e1724, doi:10.3791/1724 (2010).

    Abstract

    Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5.

    Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.

    We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

    Protocol

    1. PMN Isolation from human blood

    Use about 24 ml human blood with EDTA or Heparin (10 U/ml) as anticoagulant.

    1. Add 6 ml Histopaque 1119 to a 15 ml Falcon tube and carefully layer 5 to 7 ml whole blood on top.
    2. Centrifuge for 20 minutes at 800 x g without braking.
    3. Aspirate and discard yellowish and clear top layer and transfer lower reddish phase containing granulocytes into fresh Falcon tubes.
    4. Wash cells by filling up Falcon tubes with PBS and centrifuge for 10 minutes at 300 x g.
    5. In the meantime prepare a 100 % Percoll solution by mixing 18 ml Percoll with 2 ml 10x PBS. With 1x PBS prepare 4 ml solutions of 85 %, 80 %, 75 %, 70 % and 65 % Percoll.
    6. Prepare 2 Falcon tubes with a Percoll gradient by layering 2 ml of every percentage on top of each other in decreasing order.
    7. After centrifugation remove the supernatant, combine pellets and resuspend sedimented cells in 4 ml of PBS.
    8. Carefully layer 2 ml of the resuspension onto each of the gradients.
    9. Centrifuge for 20 minutes at 800 x g without braking.
    10. After centrifugation remove top layer and most of the 65%-layer with PBMCs and collect white remaining interphases until 85%-layer into new Falcon tubes.
    11. Wash cells by filling up the Falcon tubes with PBS and centrifuge for 10 minutes at 300 x g.
    12. Remove supernatant and resuspend sedimented cells (usually >95% are PMN) in 2ml of PBS.
    13. Count cells using a hemocytometer.

    2. Activating PMNs

    1. Prepare a 24-well cell culture plate by putting a sterile 13 mm round glass cover slip (# 1,5) into each well
    2. Seed 2 x 105 cells in 500μl RPMI (containing 2 % human serum albumin) per well and incubate for 1h in CO2 incubator at 37°C.
    3. Meanwhile prepare a 600 nM PMA solution in RPMI and add to cells 100μl per well. Incubate from 15 min up to 4 h in CO2 incubator at 37°C.
    4. Fix cells in 4% (end concentration) paraformaldehyde dissolved in PBS.

    PMA serves as a positive control and is until now the most potent agent to induce NET formation. Alternatively, other stimuli or co-cultivation with pathogens can be used for NET induction.

    The respective status of NET formation can be checked while the time course is proceeding, if additional parallel samples are prepared. If a non-permeant DNA dye like Sytox Green (Invitrogen) is added to the non-fixed cells, only extracellular DNA will be detected. Since formation of new NETs is somehow impaired in the presence of Sytox, for each time point one parallel sample has to be used.

    3. NET detection by immunolabeling

    NETs are very fragile even after fixation and have to be manipulated with great care, otherwise the majority will get lost during the preparation.

    1. Carefully remove glass cover slips with attached cells from 24-well culture plate by lifting it up at the edge with a curved needle and seize it with a fine forceps. Put the cover slip upside down on a drop of PBS. This can be done on a Parafilm sheet covering a test tube stand. Wash like this 3 times for 5 min.
    2. Incubate cover slips in the same manner in a drop of 0.5 % Triton X-100 for 1 min at RT to permeabilize cells. Wash 3 times in PBS for 1 min.
    3. Prepare a humid chamber with Parafilm and a wet tissue. Lay the cover slips upside down on a drop of blocking buffer (5% donkey serum) and incubate for 30 min at 37°C.
    4. Dilute primary antibodies, e.g. ms anti Histon and rb anti Neutrophil Elastase, in blocking buffer.
    5. Transfer cover slips in the humid chamber directly from blocking buffer onto a drop of primary antibody and incubate for 1 h at 37°C.
    6. Wash 3 times for 5 min with PBS.
    7. Dilute secondary antibodies, e.g. dk anti ms Cy2 and dk anti rb Cy3, in blocking buffer.
    8. Transfer cover slips into the humid chamber onto a drop of secondary antibody and incubate for 1 h at 37°C.
    9. Wash 3 times for 5 min with PBS. Depending on your fluorescence microscopy options, stain DNA for 5 min either with Hoechst 33342 (1 μg/ml) which will need UV excitation or with Draq5 for far red excitation and wash twice with dist. water.
    10. Set a 20μl drop of Mowiol onto a glass slide and mount cover slips with cells upside down. The cells have to be between the cover slip and the slide. The drop of Mowiol will form a thin layer of homogenous thickness when the cover slip is positioned on the drop. Normally, there is no need to press the sample. If you want to use non-immersion lenses, the specimen is ready for inspection. For microscopic analysis with immersion lenses, let the specimen dry for about 1 hour until the Mowiol has solidified at the edge of the sample.

    4. Preparing NETs for Scanning Electron Microscopy (SEM)

    1. Post fix cells on the glass cover slips in 24-well plate with 2,5 % glutaraldehyde.
    2. Remove glass cover slips containing cells from 24-well culture plate and put upside down on a drop of water. Wash like this 3 times for 5 min.
    3. Transfer cover slips back into 24-well cell culture plate containing 0,5% OsO4 and incubate for 30 min.
    4. Remove glass cover slips containing cells from 24-well culture plate and put upside down on a drop of water. Wash like this 3 times for 5 min.
    5. Transfer cover slips back into 24-well cell culture plate containing 1% tannic acid and incubate for 30 min.
    6. Repeat steps 4.2 to 4.4
    7. Dehydrate through the following regimen (5min each):
      • 30 % ethanol
      • 50 % ethanol
      • 70 % ethanol
      • 80 % ethanol
      • 90 % ethanol
      • 100% ethanol
      • 100% ethanol
      • 100% ethanol
    8. Transfer cover slips into critical point dryer and dry samples.
    9. Coat surface of specimen with 5nm platin/carbon layer using thin layer evaporator

    5. Representative Results:

    The isolation method usually yields unstimulated viable neutrophils with a purity greater than 95%. When fixed at different time points after stimulation, the immunostaining protocol shows the sequence of morphological changes during NETosis cell flattening, loss of nuclear lobules, loss of granule and nucleus integrity which leads to an increasing overlap of nuclear (i.e. histone) and granular (i.e. Neutrophil Elastase) staining. This protocol can serve as a starting point to analyze the specific interactions of pathogens with neutrophils. This interaction can be dissected in greater detail using the preparation protocol for Scanning Electron Microscopy.

    NET Fluorescence

    Sample of stimulated neutrophils stained for NET components (blue = DNA, red = histone, green = Neutrophil Elastase). The images show, besides the NET localization, the nuclear localization of DNA and histones and the granular pattern for Neutrophil Elastase.

    SEM PMA stimulation

    Scanning electron micrograph showing non stimulated and PMA-stimulated neutrophils. After stimulation, the neutrophils flatten out and produce NETs.

    SEM NETs and Shigella

    Higher resolution SEM image of Shigella bacteria trapped in NETs.

    Discussion

    The provided protocol will allow the isolation of non stimulated neutrophils at considerable purity, the induction of NET formation and the analysis of morphological changes during NETosis. When handling the specimens with care, i.e. avoiding harsh washing conditions which will result in the loss of most of the loosely attached NETs, the amount of NET formation under different stimulation conditions (duration, stimulus) can be compared. In this respect, the provided protocols can serve as a starting point to establish methods to analyze more sophisticated scenarios: neutrophil/pathogen interactions, sequence of stimuli, interplay with other immune cells.

    Disclosures

    References

    1. Brinkmann, V. Neutrophil extracellular traps kill bacteria. Science. 303, 1532-1535 Forthcoming.
    2. Urban, C. F., Reichard, U., Brinkmann, V., Zychlinsky, A. Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms. Cell Microbiol. 8, 668-676 (2006).
    3. M, E. Leishmania amazonensis promastigotes induce and are killed by neutrophil extracellular traps. PNAS. 106, 6748-6753 (2009).
    4. Buchanan, J. T., Simpson, A. J., Aziz, R. K., Liu, G. Y., Kristian, S. A., Kotb, M., Feramisco, J., Nizet, V. DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps. Curr Biol. 14, 396-400 (2006).
    5. Fuchs, T. A. Novel cell death program leads to neutrophil extracellular traps. J Cell Biol. 176, 231-241 (2007).
    6. Brinkmann, V., Zychlinsky, A. Beneficial suicide: why neutrophils die to make NETs. Nat Rev Microbiol. 5, 577-582 (2007).
    7. Aga, E., Katschinski, D. M., van Zandbergen, G., Laufs, H., Hansen, B., Muller, K., Solbach, W., Laskay, T. Inhibition of the spontaneous apoptosis of neutrophil granulocytes by the intracellular parasite Leishmania major. J Immunol. 169-898 (2002).

    Comments

    22 Comments

    Hi,
    The protocol looks good for imaging- 1) how can you be sure if you are isolating only NETs but not also neutrophil ²) how intact are the NETs? 3) Is there a particular protocol for isolating only NETs? if not how hard is it?
    Reply

    Posted by: AnonymousJune 16, 2010, 8:19 AM

    Hi - the protocol is about isolating neutrophils and induce NET formation, not about NET isolation. If you fix NETs in the supernatant of stimulated neutrophils they are pretty intact. There is a protocol for isolation and quantification of NETs in Fuchs et al.
    Reply

    Posted by: Volker B.September 1, 2010, 11:02 AM

    Hi! good day... how i can download this video? would you tell about this? i need to explain to my students...
    Thanks!
    Reply

    Posted by: AnonymousAugust 31, 2010, 3:14 PM

    Please send us an email to support@jove.com
    Reply

    Posted by: AnonymousAugust 31, 2010, 3:23 PM

    Sirs,
    This is a wonderful video on a hot subject. I will give a talk on our own work on NETS, and I would like to show the audience the way you prepare NETS. Could you permit me use your video in my talk?. If so, would you allow me to download the video?.Thank you very much.
    o. rojas-espinosa (rojas_espinosa@hotmail.com)
    Reply

    Posted by: AnonymousSeptember 3, 2010, 9:06 PM

    Hello from Greece,
    I am trying to follow your protocol and althought Imanaged to isolate the PMNs when I am trying to activate them with the pma I get results similar to the control group. I noticed that sytox has a toxic effect on the PMNs. Could you please tell me what is the concentration you are using? I would also like to ask you what is the Human Albumin serum you are using (cat no. and putity) and if you think I could use BSA cell culture tested instead.

    Ariana Gavriil, Ph.D.
    Immunology and Transplantation Center
    Foundation for Biomedical Research
    4 Soranou Ephessiou, 115 ²7
    Athens, Greece
    Tel.: +30²10 65 97 335, Fax.: +30²10 65 97 348
    Reply

    Posted by: AnonymousApril 15, 2011, 6:48 AM

    try to use 100nM of PMA from Molecular Probes
    Reply

    Posted by: AnonymousJuly 1, 2011, 5:38 PM

    What a great video. Thank you very much. The idea to present video protocols is wonderful. Unfortunately, my university has no subscription. It seems to be too expensive. I think these kind of videos would save a lot of money being spent in unseccessful efforts. :)

    Do you think that NETs can bee seen in cytospins?
    After fixation of cells in Formalin or Methanol, what would be the reason for the fragility of NET structures?
    Reply

    Posted by: AnonymousFebruary 17, 2012, 6:55 AM

    Hello all,
    I am wanting to properly prepare and perform immunostaining (using primary and secondary antibodies) on a pus-filled clinical sample to determine the presence of neutrophil nets, complete with (hopefuly) organisms trapped/attached of "caught in the act" of biofilm growth. If anyone would be so kind, please share some insight on how to proceed with this idea. I know that I will have to be careful during collection and slide preparation as to keep any nets intact, and I know I will have to fix the slides somehow before I start the staining process. Everything else is beyond my knowledge. Any ideas on how to proceed?
    Reply

    Posted by: AnonymousMarch 3, 2012, 11:06 AM

    Dear colleagues, thank you very much for sharing the experience.
    Could you tell me where did you obtain H²A-H²B-DNA complex antibody (they seems to be not commercial) or recommend some other anti Histone antibodies?
    Reply

    Posted by: AnonymousMarch 7, 2012, 8:17 AM

    This is a novice Percoll user question. I prepared the percoll solutions with PBS as described under "1. PMN Isolation from human blood" step number 5. I made large enough volumes to prepare multiple gradients over a period of several days, and the solutions were stored @ 4C until needed. The first two neutrophil separations worked fine in that the RBCs pelleted as expected. The last two times resulted in incomplete pelleting of the RBCs leaving some left mixed in with the neutrophils. Should I be making fresh percoll solutions each time? Any other suggestions?
    Reply

    Posted by: M C.September 24, 2012, 2:29 PM

    It is OK to store made up Percoll solutions at 4°C for some days or even weeks and the fact of storing them alone should not cause incomplete separation of the RBCs. The lowest band of PMN just above the RBC pellet often contains a larger portion of RBCs and can be omitted if you don't need the highest possible PMN count. If the RBC pellet dŒsn't form clearly at all there might still be something wrong with your solutions or the exact layering of the discontinuous gradient. First, try running the tubes longer to see if sedimentation was just slower than normal. There could also be causes in the preceding steps, so you might have to do more troubleshooting.
    Reply

    Posted by: Christian G.September 25, 2012, 4:37 AM

    Thank you for your quick response and suggestions!
    Reply

    Posted by: M C.September 25, 2012, 8:20 AM

    Thanks for the great video! In which step (if any) would it be possible to stop the protocol and continue the next day? Perhaps after fixing with the formaldehyde I could wash and store them overnight in PBS at 4C. I am trying to visualize NETs from mouse neutrophils collected from the bone marrow and just purifying the PMN takes half a day.
    Reply

    Posted by: Xavier R.November 21, 2012, 12:23 PM

    Hi,thanks for your great video! It's very useful for me. I want to know whether the cover slips were lysinated before incubated neutrophils. Thank you !
    Reply

    Posted by: Yin X.June 25, 2013, 9:00 AM

    We think it works best on glass slips that were not treated with polylysin.
    Reply

    Posted by: Christian G.June 25, 2013, 11:25 AM

    But ,if not,how did neutrophils seed at slips stabe since they were not adherent cell?
    Reply

    Posted by: Yin X.June 25, 2013, 11:34 AM

    Dear sir:
    For my experiment I am trying to isolate mature Neutrophils from peritoneal cavity of mice.
    Can you help me to answer my few questions please.

    How to prepare 3% thioglycollate broth?
    how many hours we should wait after injecting 3% thioglycollate to get mature neutrophils?

    Thank you
    Piya Patel
    Reply

    Posted by: Piya P.January 17, 2014, 11:54 AM

    hi,

    Thank you for the protocol :) it is well explain !
    Have questions tough :
    -for fixation : you put the PFA within the media or do you first take it out then rinse with PBS then fix ?
    - can I fix and freeze the coverslip at -80°C to do the staining later ?
    - can I use mounting media with DAPI instead of staining DAPI then mounting ?

    Thank you very much !
    Anthony
    Reply

    Posted by: anthony l.May 6, 2014, 12:56 PM

    Hi Anbthony,

    We put double concentrated PFA directly into the warm medium. Rinsing would destroy most of the NETs. Onced the specimens are fixed you can keep them for some time until staining. We never freeze them but keep them refrigerated after replacing the fixative with PBS (gently!). You can use DAPI-containing embedding media but we get less background when we wash the cells after DAPI staining.

    Good luck, Volker
    Reply

    Posted by: Volker B.May 7, 2014, 9:52 AM

    Hi,

    Thank for the tips !
    It worked beautifully ;).
    I fixed with PFA as you explain, then wash 1x pbs carrefully Freeze them at -80°C and used mouting media with DAPI.
    PMA use : 600nM for 1h at 37°C.

    Thanks!
    anthony
    Reply

    Posted by: anthony l.May 15, 2014, 8:22 AM

    Hi, I have a few questions regarding the isolation.
    Is it possible to isolate PMN's from buffy coat or is it only possible do this from Whole blood?
    How many PMN's is it possible to get if following this protocol? How much blood did you start with?
    Is it important to use 15 mL tubes when using both Histopaque and Percoll, or does it work with 50 mL tubes?

    Thank you in advance!
    Helena

    Reply

    Posted by: Helena E.May 20, 2014, 9:31 AM

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