This work was supported by the Fondation Laurette Fugain (LF-2015-15) (B.S.M.) and ANR JCJC grants (ANR-17-CE15-0012) (B.S.M.).

All methods described here have been reviewed and approved by the French organizations CoRC (Comit&#233; de Recherche Clinique), CPP (Comit&#233; de Protection des Personnes), and CNIL (Commission Nationale Informatique et Libert&#233;).
1. Staining Neutrophils in Histopathology Tissue with Fluorescently Labelled MUB40
<ol>
	<li>To obtain the tissue for histopathology, fix tissue/biopsy sections in a suitable volume of 4% paraformaldehyde (PFA) for 30 min up to 4 h depending on the thickness of the sample. Place the samples in a 4 &#176;C refrigerator during fixation. 
	Note: Alternative fixation methods/timings can be substituted based on the user&#39;s unique needs.
	<ol>
		<li>Remove the PFA, add 16% sucrose solution with enough volume to completely cover the sample, and place the sample at 4 &#176;C for 4 h up to overnight.</li>
		<li>Remove 16% sucrose solution and add 30% sucrose solution at a large enough volume to completely cover the sample, and place it at 4 &#176;C for 4 h up to overnight.</li>
		<li>Remove the 30% sucrose solution and blot off excess solution from the tissue with a paper towel. Place the tissue section in optimal cutting temperature (OCT) media and snap-freeze in 2-methylbutane cooled in a dry ice-ethanol bath (-72 &#176;C). Once frozen solid (after ~2 min), remove blocks from the 2-methylbutane and place them on dry ice until all blocks are finished and ready for long-term storage. Store the frozen blocks at -80 &#176;C for long-term storage.</li>
	</ol>
	</li>
	<li>The night before tissue blocks are to be cut, remove them from the -80 &#176;C freezer and place them at -20 &#176;C overnight to equilibrate.
	<ol>
		<li>Using a cryostat, cut OCT-embedded tissue into 10- to 30 &#956;m-thick slices and adsorb to high-quality glass slides. 
		Note: Thicker or thinner slices can be used depending on the experimental requirements.</li>
		<li>With a wax pen or other hydrophobic method, draw a box around the tissue slice on the glass slide and let dry.</li>
		<li>Prepare the staining solution by performing a 1:1,000 dilution of MUB40-Cy5 (or other fluorescent MUB40version, 1 mg/mL stock solution) in 0.1% saponin-PBS solution. The optimal final MUB40 concentration should be 1 &#956;g/mL. 
		Note: Lower final concentrations may be used (0.1-0.01 &#956;g/mL) but may lead to lower signal intensity. 
		CAUTION: Saponin powder can cause breathing irritation. Make sure to weigh saponin in a cabinet or protected area. Alternative permeabilization methods can be used such as Triton. Add additional markers at manufacturer-recommended concentrations (e.g., 4&#8242;,6-diamidino-2-phenylindole (DAPI), phalloidin, fluorescent antibodies).</li>
		<li>Gently dip the glass slide into a solution of 0.1% saponin-PBS three times.</li>
		<li>Carefully blot away excess liquid around the tissue section and add enough staining solution to completely cover the tissue section. Place the slide into a humidity chamber to avoid evaporation and incubate it for 1 h at room temperature. Keep it protected from light.</li>
		<li>Gently dip the slide into 0.1% saponin-PBS solution 3x. Then, gently dip the slide into PBS solution 3x. Finally, gently dip the slide into distilled H2O 3x. Carefully dry excess liquid from the slide.</li>
		<li>Add a small amount (~20 &#956;L) of mounting medium (20 mM Tris pH 8.0, 0.5% N-propyl gallate, 90% glycerol) next to the tissue slice and gently lay down a glass coverslip on top of the glass slide. Gently and evenly press the coverslip onto the tissue section and, using a paper towel, carefully remove any mounting media that extrudes around the edges of the coverslip.</li>
		<li>Place the mounted slide in a dark cabinet for 24 h to allow the mounting media to solidify. Alternatively, place the mounted slide at 37 &#176;C for 1 h.</li>
	</ol>
	</li>
	<li>Image the tissue slices on a fluorescent microscope according to the desired acquisition method.</li>
</ol>
2. Visualization of Neutrophil Activation with Retro-Inverso-MUB40-Cy5 Peptide
<ol>
	<li>Obtain human neutrophils using the following protocol. Prepare the following solutions and place them in an anoxic cabinet overnight. Exposure to oxygen will begin to activate neutrophils and further induce cell death16,17. 
	Note: Neutrophils may be alternatively purified &#34;on the bench&#34; for MUB40-labeling experiments; however, be aware that oxygen exposure will begin to affect neutrophil activation.
	<ol>
		<li>Prepare the following solution: 
		Solution 1 [sodium chloride (NaCl) 0.9%]: add 4.5 g of NaCl to 500 mL of H2O. Filter the solution. 
		Solution 2 (dextran 6%): add 6 g of dextran in NaCl 0.9% solution to 100 mL. 
		Solution 3 (washing buffer): dissolve ethylenediaminetetraacetic acid (EDTA) in PBS pH 7.2 to a final EDTA concentration of 2 mM. Immediately prior to use, dissolve bovine serum albumin (BSA) in the PBS/EDTA solution such that the final BSA concentration is 10% w/v.</li>
		<li>Centrifuge blood collection tubes at 650 x g for 20 min without breaks.</li>
		<li>Without disrupting separated blood, transfer the blood collection tubes to an anoxic cabinet and collect plasma fractions (top) in a 50 mL conical tube.</li>
		<li>Remove the plasma-containing tube from the anoxic cabinet and centrifuge at 2,900 x g for 20 min with breaks to pellet the platelets.</li>
		<li>Without disrupting the pelleted platelets, gently transfer the plasma tube back into the anoxic cabinet and pipet the platelet poor plasma into a fresh 50 mL tube (hereafter called &#34;plasma&#34;).</li>
	</ol>
	</li>
	<li>Separation of red blood cells from leukocytes
	<ol>
		<li>Using the tubes containing the red blood cells (RBCs) from step 2.1.3., combine the RBCs into a conical 50 mL tube (5 blood collection tube contents per 50 mL tube).</li>
		<li>Add NaCl 0.9% until the total volume reaches 44 mL. Add 6 mL of dextran 6% (last).</li>
		<li>Gently mix the blood/dextran mixture by inverting the tube 10-20 times. Let the tube sediment for at least 30 min.</li>
		<li>While sedimentation is occurring, prepare a Percoll gradient by adding 4.2 mL of Percoll solution to 5.8 mL of plasma in a 15 mL conical tube, and mix well by inverting the tube.</li>
		<li>Collect the top fraction of the dextran while trying to avoid pipetting red blood cells.</li>
		<li>Tighten the screw cap, remove the dextran tube from the anoxic chamber, and centrifuge the tube at 300 x g for 10 min with breaks.</li>
		<li>Carefully place the tube back into the anoxic chamber without disturbing the pelleted cells, and remove the liquid via pipetting.</li>
		<li>Gently re-suspend the cell pellet in 1 mL of plasma and very slowly add to the top of the Percoll solution. Be careful not to induce mixing of the layers when pipetting the cells on top.</li>
		<li>Carefully remove the Percoll solution tube from the anoxic chamber (avoiding mixing), and centrifuge at 800 x g for 20 min without breaks. Peripheral blood mononuclear cells (PBMCs) will remain on the Percoll solution surface, and neutrophils will pellet with the RBCs.</li>
	</ol>
	</li>
	<li>Removal of contaminating RBCs
	<ol>
		<li>Prepare 14 mL of washing buffer solution by adding 700 &#956;L PBS + 10% BSA to 14 mL of washing buffer.</li>
		<li>Place the Percoll solution tube back into the anoxic chamber. Remove the Percoll and PBMCs via pipetting and re-suspend the pelleted cells in 1 mL of washing buffer solution. The pellet to be re-suspended will have a red color due to contaminating RBCs.</li>
		<li>Add 200 &#956;L of anti-CD235a (glycophorin) magnetic beads to the re-suspended cells, gently mix, and incubate for a minimum of 15 min. This step loads the contaminating RBCs with magnetic beads so that they can be removed.</li>
		<li>Wash the separation column with 2 mL of washing buffer 3x.</li>
		<li>While holding a 15 mL conical tube under the separation column, slowly pipette the neutrophil/RBC mixture to the top of the column. Collect the cells in the 15 mL conical tube as they flow through. The flow-through should be cloudy and lose its red color due to the RBCs remaining bound to the column.</li>
		<li>Add 2 mL of washing buffer to the top of the column and collect in the same 15 mL conical tube. By the end of the 2 mL wash, the flow-through should be clear, signifying that all the neutrophils have been collected.</li>
		<li>Gently mix the tube to ensure even distribution of neutrophils and collect 10 &#956;L for cell counting. Note the final volume for cell counting purposes. Enumerate the total number of cells with any standard cell counting method.</li>
		<li>Remove the neutrophil tube from the anoxic chamber, and centrifuge the neutrophils at 300 x g for 20 min with breaks.</li>
		<li>Place sedimented neutrophils back into the anoxic chamber and remove the washing buffer via pipetting. Re-suspend the neutrophil pellet in plasma with a maximum cell density of 107&#160;PMN/mL.</li>
	</ol>
	</li>
	<li>Enumeration of purified neutrophils and visualization using MUB40
	<ol>
		<li>Add 1 &#956;L of RI-MUB40-Cy5 (1mg/mL) to 1 mL of Rosewell Park Memorial Institute (RPMI) 1640 medium without phenol red. Mix by pipetting well.</li>
		<li>For the purposes of this protocol, the result has been outlined as if a potent neutrophil activating reagent, N-formylmethionyl-leucyl-phenylalanine (FMLP), is added. Add 1 &#956;m of FMLP to the RPMI/RI-MUB40-Cy5 tube and mix via pipetting up and down. 
		NOTE: Each experimental procedure will be different from this point forward, depending on the questions being addressed.</li>
		<li>Transfer the 1 mL RPMI/RI-MUB40-Cy5/FMLP mixture to a glass bottom microscopy dish. To this dish, add a volume of purified cells corresponding to 106&#160;total neutrophils. Mix by gently pipetting.</li>
		<li>Place the dish into an inverted fluorescent microscope and start image acquisition. For example, it is recommended to acquire baseline images prior to the addition of a neutrophil-activating reagent such as FMLP or bacteria. In this example, start the acquisition of RPMI/RI-MUB40-Cy5/neutrophils, and at a later timepoint, pipette the neutrophil-activating reagent into the glass dish and continue image acquisition. 
		NOTE: Modifications to these steps can be made based on scientific needs.</li>
		<li>Process acquired images/time-lapse movies as pertains to the specific microscope used.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Nathan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+and+immunity:+challenges+and+opportunities.">Neutrophils and immunity: challenges and opportunities.</a> Nature Reviews Immunology. 6 (3), 173-182 (2006).</li><li>Nicol&#225;s-&#193;vila, J. &. #. 1. 9. 3. ;., Adrover, J. M., Hidalgo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+in+Homeostasis,+Immunity,+and+Cancer.">Neutrophils in Homeostasis, Immunity, and Cancer.</a> Immunity. 46 (1), 15-28 (2017).</li><li>Coffelt, S. B., Wellenstein, M. D., de Visser, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+in+cancer:+neutral+no+more.">Neutrophils in cancer: neutral no more.</a> Nature Reviews Cancer. 16 (7), 431-446 (2016).</li><li>Wright, H. L., Moots, R. J., Bucknall, R. C., Edwards, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+function+in+inflammation+and+inflammatory+diseases.">Neutrophil function in inflammation and inflammatory diseases.</a> Rheumatology. 49 (9), 1618-1631 (2010).</li><li>Glennon-Alty, L., Hackett, A. P., Chapman, E. A., Wright, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+and+redox+stress+in+the+pathogenesis+of+autoimmune+disease.">Neutrophils and redox stress in the pathogenesis of autoimmune disease.</a> Free Radical Biology &amp; Medicine. , (2018).</li><li>Borregaard, N., Cowland, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granules+of+the+human+neutrophilic+polymorphonuclear+leukocyte.">Granules of the human neutrophilic polymorphonuclear leukocyte.</a> Blood. 89 (10), 3503-3521 (1997).</li><li>Borregaard, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils,+from+marrow+to+microbes.">Neutrophils, from marrow to microbes.</a> Immunity. 33 (5), 657-670 (2010).</li><li>Rose, S., Misharin, A., Perlman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+Ly6C/Ly6G-based+strategy+to+analyze+the+mouse+splenic+myeloid+compartment.">A novel Ly6C/Ly6G-based strategy to analyze the mouse splenic myeloid compartment.</a> Cytometry Part A: The Journal of the International Society for Analytical Cytology. 81 (4), 343-350 (2012).</li><li>Shi, C., Hohl, T. M., Leiner, I., Equinda, M. J., Fan, X., Pamer, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ly6G++neutrophils+are+dispensable+for+defense+against+systemic+Listeria+monocytogenes+infection.">Ly6G+ neutrophils are dispensable for defense against systemic Listeria monocytogenes infection.</a> Journal of Immunology. 187 (10), 5293-5298 (2011).</li><li>Albanesi, M., Mancardi, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+mediate+antibody-induced+antitumor+effects+in+mice.">Neutrophils mediate antibody-induced antitumor effects in mice.</a> Blood. 122 (18), 3160-3164 (2013).</li><li>Anderson, M. C., Chaze, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MUB40+Binds+to+Lactoferrin+and+Stands+as+a+Specific+Neutrophil+Marker.">MUB40 Binds to Lactoferrin and Stands as a Specific Neutrophil Marker.</a> Cell Chemical biology. 25 (4), 483-493 (2018).</li><li>Co&#239;c, Y. -. M., Baleux, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+a+specific+colonic+mucus+marker+using+a+human+commensal+bacterium+cell+surface+domain.">Design of a specific colonic mucus marker using a human commensal bacterium cell surface domain.</a> Journal of Biological Chemistry. 287 (19), 15916-15922 (2012).</li><li>Roos, S., Jonsson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-molecular-mass+cell-surface+protein+from+Lactobacillus+reuteri+1063+adheres+to+mucus+components.">A high-molecular-mass cell-surface protein from Lactobacillus reuteri 1063 adheres to mucus components.</a> Microbiology. 148, 433-442 (2002).</li><li>Boekhorst, J., Helmer, Q., Kleerebezem, M., Siezen, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+proteins+with+a+mucus-binding+domain+found+exclusively+in+lactic+acid+bacteria.">Comparative analysis of proteins with a mucus-binding domain found exclusively in lactic acid bacteria.</a> Microbiology. 152, 273-280 (2006).</li><li>Mancardi, D. A., J&#246;nsson, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+Edge:+The+murine+high-affinity+IgG+receptor+Fc&#947;RIV+is+sufficient+for+autoantibody-induced+arthritis.">Cutting Edge: The murine high-affinity IgG receptor Fc&#947;RIV is sufficient for autoantibody-induced arthritis.</a> Journal of Immunology. 186 (4), 1899-1903 (2011).</li><li>Walmsley, S. R., Print, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-induced+neutrophil+survival+is+mediated+by+HIF-1alpha-dependent+NF-kappaB+activity.">Hypoxia-induced neutrophil survival is mediated by HIF-1alpha-dependent NF-kappaB activity.</a> Journal of Experimental Medicine. 201 (1), 105-115 (2005).</li><li>Monceaux, V., Chiche-Lapierre, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anoxia+and+glucose+supplementation+preserve+neutrophil+viability+and+function.">Anoxia and glucose supplementation preserve neutrophil viability and function.</a> Blood. 128 (7), 993-1002 (2016).</li></ol>

B.S.M. is listed as an inventor on MUB40&#160;patents:
MUB40: European Patent EP11290403.2, 09/09/2011.
RI-MUB40: European Patent EP no. 17306746.3, 11/12/17.

Here, two assays are described in which the MUB40&#160;peptide is used to study neutrophil inflammation and activation. We show how MUB40&#160;can reveal neutrophils present in histopathology sections or show their activation state using live purified neutrophils. The critical steps for using MUB40 as a staining reagent are the same as with any other fluorescent detection method. Care must be taken to ensure compatibility of fluorescent signals and that adequate washing steps have been ta...

Neutrophils are one of the primary arms of the innate immune system and are routinely recruited to the sites of inflammation around the body. The study of neutrophils has been largely impaired by their short lifespan in vitro (less than 8 h) and by limited detection tools under basal conditions or after activation. Here, we present a well-tested protocol for the broad and specific detection of mammalian neutrophils in fixed/permeabilized samples. We also provide a detailed protocol for staining live neutrophils with MUB40. Using the live neutrophil staining protocol, the timing and location of neutrophil activation can be pinpointed. This protocol is ideal for researchers who wish to study neutrophil activation or granule release. Beyond their antimicrobial functions, neutrophils are now appreciated as immunomodulatory cells involved in a wide range of diseases and immune responses (innate and adaptive)1,2. Neutrophils are present in most inflammatory tissues, at high numbers in infected tissues, in inflammatory tumors3, during IBS and Crohn&#39;s flare-ups4, and in areas of non-infectious inflammation such as the synovia of Rheumatoid Arthritis (RA) patients5. Neutrophils contain four classes of pre-formed granules named azurophil (&#945;), specific (&#946;1), tertiary (&#946;2) granules, and secretory vesicles (&#947;)6. Upon migration to an inflammatory site, recruited neutrophils become activated and sequentially secrete granule contents (composed of adhesion, antimicrobial compounds and immunomodulatory molecules), which promotes further recruitment and contributes to infection resolution (but also to host tissue damage)7. While neutrophils are considered a critical aspect of innate immunity, to date there are few detection reagents available to study them, and even fewer that can be used to assess the activation state of living neutrophils.
Current methods of detecting neutrophils rely on monoclonal antibodies generated against cell-surface exposed antigens, such as Ly-6G2&#160;or proteins specifically stored in neutrophil granules under basal conditions (e.g., myeloperoxidase, lactoferrin). Advantages of monoclonal antibodies include their strong binding, sensitivity, and versatility under various assay conditions. However, there are several downsides to using monoclonal antibodies and anti-Ly-6G in particular. These downsides include the specificity, as Ly-6G is present on a majority of myeloid cells in bone marrow and on all granulocytes including eosinophils; thus, deciphering neutrophils from eosinophils with this marker requires more complexes approaches8. Another frequent tradeoff with monoclonal antibodies is their often-limited host specificity range, making comparison studies with more than one animal species difficult. A third drawback of antibody detection methods, especially when using live cells or in vivo, is their potential to disrupt cell function or lead to cell activation. For example, the administration of anti-Ly-6G to mice is commonly applied to neutrophil depletion and transient neutropenia9. It has additionally been demonstrated that antibody injection may stimulate neutrophil antitumor function10. Finally, antibody detection of neutrophils does not reveal the activation state of the cell.
We have identified a 40-amino acid peptide called MUB40, which can be used in a number of assays to label neutrophils under in vitro or in vivo conditions as well as assay the activation status of live neutrophils11. MUB40&#160;is derived from MUB7012, a domain of the Lactobacillus reuteri surface mucus-binding protein originally described for its ability to bind mucus13,14. MUB40&#160;interacts with glycosylated lactoferrin that is present at high concentrations in neutrophil-specific and tertiary granules. MUB40 can be exposed to these granules through standard permeabilization steps present in histopathology or fluorescence-activated cell sorting (FACS) protocols for robust staining of fixed neutrophils. When live neutrophils are kept in an inactivated state, the MUB40&#160;peptide is excluded from the granule contents, and cells do not stain positive with the marker. Upon activation, MUB40&#160;can bind to exposed lactoferrin and lead to robust staining with the peptide. Thus, MUB40 can be used to determine the activation state of purified neutrophils, making it an attractive marker to follow the infectious process. The ability to bind to live activated neutrophils also allows MUB40&#160;to be used as a tool to detect areas of neutrophil inflammation in live animal models (e.g., a mouse arthritis model15). The protocols in this manuscript detail how fluorescently labeled MUB40 can be used to reveal neutrophils in histology tissues and how to assay the activation of live purified neutrophils in vitro.

<table><tbody><tr><td>MUB&lt;sub&gt;40&lt;/sub&gt;-Cy5</td><td>Benoit Marteyn</td><td>benoit.marteyn@pasteur.fr</td><td>Fluorescent MUB&lt;sub&gt;40&lt;/sub&gt; peptide (available with other conjugated fluorophores)</td></tr><tr><td>RI-MUB&lt;sub&gt;40&lt;/sub&gt;-Cy5</td><td>Benoit Marteyn</td><td>benoit.marteyn@pasteur.fr</td><td>Retro-inverso fluorescent MUB40 peptide. Synthesized with D-amino acids and resistant to proteases (available with other conjugated fluorophores)</td></tr><tr><td>Parformaldehyde</td><td>Sigma-Aldrich</td><td>16005</td><td>Fixative for histology</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S7903</td><td>Solution used to remove excess PFA</td></tr><tr><td>Optimal Cutting Temperature Compound (OCT)</td><td>Sakura Finetek USA</td><td>4583</td><td>Used to freeze tissue before cryostat sectioning</td></tr><tr><td>2-Methylbutane</td><td>Sigma-Aldrich</td><td>M32631</td><td>Used to freeze tissue before cryostat sectioning</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>1.00974</td><td>Used for dry ice bath to freeze tissue</td></tr><tr><td>Leica Cryostat</td><td>Leica Biosystems</td><td>CM1520</td><td>Used to section tissues</td></tr><tr><td>Glass microscopy slides</td><td>Fisher Scientific</td><td>12-518-101</td><td></td></tr><tr><td>Cover slips</td><td>Thor Labs</td><td>CG15CH</td><td>Hi precision coverslip</td></tr><tr><td>Dako pen&amp;nbsp;</td><td>Sigma-Aldrich</td><td>Z377821</td><td>Used to prevent liquid loss during tissue staining</td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>47036</td><td>Used to permeabilize cells for IF staining</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>10236276001</td><td>Stains DNA&amp;nbsp;</td></tr><tr><td>Alexa fluor 488 Phalloidin&amp;nbsp;</td><td>Fisher Scientific</td><td>A12379</td><td>Binds actin</td></tr><tr><td>Prolong Gold antifade Mountant</td><td>Fisher Scientific</td><td>P36930</td><td>Prolongs IF signal and resistance to photobleaching</td></tr><tr><td>Fluorescent microscope</td><td>Various</td><td>Various</td><td>Used to image IF slides</td></tr><tr><td>Anoxic Cabinet</td><td>Various</td><td>Various</td><td>Used for the purification of live inactivated neutrophils</td></tr><tr><td>Sodium Chloride NaCL</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E9884</td><td>Washing buffer component</td></tr><tr><td>MACS BSA buffer</td><td>Miltenyi Biotec</td><td>130-091-376</td><td>Washing buffer component</td></tr><tr><td>Percoll</td><td>Sigma-Aldrich</td><td>P4937</td><td>Gradient for neutrophil purification</td></tr><tr><td>CD235a glycophorin magnetic microbeads</td><td>Miltenyi Biotec</td><td>130-050-501</td><td>Used to remove contaminating RBCs</td></tr><tr><td>LS columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td>Used in the removal of RBCs from neutrophils</td></tr><tr><td>RPMI-1640 without Phenol Red</td><td>Sigma-Aldrich</td><td>R8755</td><td>Used for neutrophil assays</td></tr></tbody></table>

the mub40 peptide for use in detecting neutrophil-mediated inflammation events

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high concentrations in specific and tertiary granules of neutrophils. This protocol details how MUB40&#160;conjugated directly to a fluorophore can be used to stain neutrophils in fixed/permeabilized tissues as well as how this can be used in live-cell imaging to assay for neutrophil activation and de-granulation. Neutrophil detection methods are limited to species-specific monoclonal antibodies, which are not always suitable for certain applications. MUB40 does not penetrate the cell membrane and is thus excluded from lactoferrin stored in non-activated/non-permeabilized neutrophils. MUB40 has the added benefit of recognizing lactoferrin from a broad host range, making it especially useful for comparing results in studies involving multiple research models, reducing the number of duplicate reagents, and simplifying protocols through single-step staining.

Results of MUB40-stained tissues from histopathology slides typically reveal individual cells scattered throughout the tissue. MUB40&#160;stains lactoferrin, which is present in neutrophil granules and compartmentalized. Thus, typically seen are punctate staining or several large separated areas of signal coming from individual neutrophils (Figure 1). It is helpful to add a second cell marker such as DAPI to help co-localize the MUB...

Watch this Scientific Journal Video about The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events at JoVE.com

The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

Here, we present a protocol to detect the presence of neutrophils in fixed/permeabilized histology sections and assess the activation state of live purified neutrophils. In particular, the MUB40&#160;peptide binds lactoferrin present in neutrophil-specific and tertiary granules. Exposure of the granule contents through either permeabilization or neutrophil activation allows for the marking of neutrophils.

The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high ...

the-mub40-peptide-for-use-detecting-neutrophil-mediated-inflammation

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JoVE Journal

Biology

6.0K Views.  Institut Pasteur. Here, we present a protocol to detect the presence of neutrophils in fixed/permeabilized histology sections and assess the activation state of live purified neutrophils. In particular, the MUB40 peptide binds lactoferrin present in neutrophil-specific and tertiary granules. Exposure of the granule contents through either permeabilization or neutrophil activation allows for the marking of neutrophils.

Video: The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Immunology and Infection

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. 
Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

Monitoring Neutrophil Elastase and Cathepsin G Activity in Human Sputum Samples

Proteases are regulators of countless physiological processes and the precise investigation of their activities remains an intriguing biomedical challenge. Among the ~600 proteases encoded by the human genome, neutrophil serine proteases (NSPs) are thoroughly investigated for their involvement in the onset and progression of inflammatory conditions including respiratory diseases. Uniquely, secreted NSPs not only diffuse within extracellular fluids but also localize to plasma membranes. During neutrophil extracellular trap (NETs) formation, NSPs become an integral part of the secreted chromatin. Such complex behavior renders the understanding of NSPs pathophysiology a challenging task. Here, detailed protocols are shown to visualize, quantify and discriminate free and membrane-bound neutrophil elastase (NE) and cathepsin G (CG) activities in sputum samples. NE and CG are NSPs whose activities have pleiotropic roles in the pathogenesis of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD): they promote tissue remodeling, regulate downstream immune responses and correlate with lung disease severity. The protocols show how to separate fluid and cellular fraction, as well as the isolation of neutrophils from human sputum for enzymatic activity quantification via small-molecule F&#246;rster resonance energy transfer-based (FRET) reporters. To gather specific insights into the relative role of NE and CG activities, a FRET readout can be measured by different technologies: i) in vitro plate reader measurements allow for high-throughput and bulk detection of protease activity; ii) confocal microscopy spatiotemporally resolves membrane-bound activity at the cell surface; iii) small-molecule FRET flow cytometry enables for the rapid evaluation of anti-inflammatory treatments via single-cell protease activity quantification and phenotyping. The implementation of such methods opens the doors to explore NSPs pathobiology and their potential as biomarkers of disease severity for CF and COPD. Given their standardization potential, their robust readout and simplicity of transfer, the described techniques are immediately shareable for implementation across research and diagnostic laboratories.

The protocols herein described provide a guide to visualize and quantify the activity of neutrophil proteases in human sputum. The applications of such analysis span from the evaluation of anti-inflammatory treatments, to biomarker validation, drug screening and large cohort clinical studies.

Proteases are regulators of countless physiological processes and the precise investigation of their activities remains an intriguing biomedical ...

CRISPR-Cas Synthetic Urine Biomarker Test for Cancer Diagnostics

CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for traditional biofluid measurements. Synthetic biomarkers generally make use of reporters that provide readable signals in the biofluid to reflect the biochemical alterations in the local disease microenvironment during disease incidence and progression. The pharmacokinetic concentration of the reporters and biochemical amplification of the disease signal are paramount to achieving high sensitivity and specificity in a diagnostic test. Here, a cancer diagnostic platform is built using one format of synthetic biomarkers: activity-based nanosensors carrying chemically stabilized DNA reporters that can be liberated by aberrant proteolytic signatures in the tumor microenvironment. Synthetic DNA as a disease reporter affords multiplexing capability through its use as a barcode, allowing for the readout of multiple proteolytic signatures at once. DNA reporters released into the urine are detected using CRISPR nucleases via hybridization with CRISPR RNAs, which in turn produce a fluorescent or colorimetric signal upon enzyme activation. In this protocol, DNA-barcoded, activity-based nanosensors are constructed and their application is exemplified in a preclinical mouse model of metastatic colorectal cancer. This system is highly modifiable according to disease biology and generates multiple disease signals simultaneously, affording a comprehensive understanding of the disease characteristics through a minimally invasive process requiring only nanosensor administration, urine collection, and a paper test which enables point-of-care diagnostics.

Author Spotlight: Engineering Molecular Tools for Disease Detection and Imaging

This protocol describes a CRISPR-Cas-mediated, multianalyte synthetic urine biomarker test that enables point-of-care cancer diagnostics through the ex vivo analysis of tumor-associated protease activities.

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for ...

Bioengineering

Quantifying Myeloperoxidase-DNA and Neutrophil Elastase-DNA Complexes from Neutrophil Extracellular Traps by Using a Modified Sandwich ELISA

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures composed of DNA with granule proteins, such as myeloperoxidase (MPO) and neutrophil elastase (NE), and cytoplasmic and cytoskeletal proteins. Although interest in NETs has increased recently, no sensitive, reliable assay method is available for measuring NETs in clinical settings. This article describes a modified sandwich enzyme-linked immunosorbent assay to quantitatively measure two components of circulating NETs, MPO-DNA and NE-DNA complexes, which are specific components of NETs and are released into the extracellular space as breakdown products of NETs. The assay uses specific monoclonal antibodies for MPO or NE as the capture antibodies and a DNA-specific detection antibody. MPO or NE binds to one site of the capture antibody during the initial incubation of samples containing MPO-DNA or NE-DNA complexes. This assay shows good linearity and high inter-assay and intra-assay precision. We used it in 16 patients with COVID-19 with accompanying acute respiratory distress syndrome and found that the plasma concentrations of MPO-DNA and NE-DNA were significantly higher than in the plasma obtained from healthy controls. This detection assay is a reliable, highly sensitive, and useful method for investigating the characteristics of NETs in human plasma and culture supernatants.

We present a protocol for a modified sandwich enzyme-linked immunosorbent assay technique to quantitatively measure two components of neutrophil extracellular trap remnants, myeloperoxidase conjugated-DNA and neutrophil elastase conjugated-DNA complexes,derived from activated neutrophils.

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures ...

Neutrophil Functional Assays for Screening Different Signaling Pathways

A Set of Screening Techniques for a Quick Overview of the Neutrophil Function

Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as chemotaxis, reverse migration, phagocytosis, degranulation of cytotoxic enzymes and metabolites, and release of DNA as neutrophil extracellular traps (NETs). Neutrophils not only have tightly regulated signaling themselves, but also participate in the regulation of other components of the immune system. As fresh neutrophils are terminally differentiated, short-lived, and highly variable among individuals, it is important to make the most of the collected samples. Researchers often need to perform screening assays to assess an overview of the many neutrophil functions that may be affected by specific conditions under evaluation. A set of tests following a single isolation process of normal density neutrophils was developed to address this need, seeking a balance between speed, comprehensiveness, cost, and accuracy. The results can be used to reason and guide in-depth follow-up studies. This procedure can be carried out in an average time of 4 h and includes the evaluation of cell viability, reactive oxygen species (ROS) production, real-time migration, and phagocytosis of yeast on glass slides, leaving enough cells for more detailed approaches like omics studies. Moreover, the procedure includes a way to easily observe a preliminary suggestion of NETs after fast panoptic staining observed by light microscopy, with a lack of specific markers, albeit enough to indicate if further efforts in that way would be worthwhile. The diversity of functions tested combines common points among tests, reducing the analysis time and expenses. The procedure was named NeutroFun Screen, and although having&#160;limitations, it balances the aforementioned factors. Furthermore, the aim of this work is not a definite test set, but rather a guideline that can easily be adjusted to each lab&#39;s resources and demands.

Author Spotlight: Balancing Speed, Cost, and Accuracy in Neutrophil Function Assessment with NeutroFun Screen Protocol 

This protocol features a set of neutrophil functional assays to be used as a screening method to cover functions from different signaling pathways. The protocol includes an initial and simple evaluation of cell viability, purity, reactive oxygen species production, real-time migration, phagocytosis, and a preliminary suggestion of neutrophil extracellular traps.

Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as ...

Detecting Migration and Infiltration of Neutrophils in Mice

Neutrophils are a major member of the innate immune system and play pivotal roles in host defense against pathogens and pathologic inflammatory reactions. Neutrophils can be recruited to inflammation sites via the guidance of cytokines and chemokines. Overwhelming infiltration of neutrophils can lead to indiscriminate tissue damage, such as in rheumatoid arthritis (RA). Neutrophils isolated from peritoneal exudate respond to a defined chemoattractant, N-formyl-Met-Leu-Phe (fMLP), in vitro in Transwell or Zigmond chamber assays. The air pouch experiment can be used to evaluate the chemotaxis of neutrophils towards lipopolysaccharide (LPS) in vivo. The adjuvant-induced arthritis (AA) mouse model is frequently used in RA research, and immunohistochemical staining of joint sections with anti-myeloperoxidase (MPO) or anti-neutrophil elastase (NE) antibodies is a well-established method to measure neutrophil infiltration. These methods can be used to discover promising therapies targeting neutrophil migration.

Here, we present three methods to assess neutrophil migration and infiltration both in vivo and in vitro. These methods can be used to discover promising therapeutics targeting neutrophil migration.

Neutrophils are a major member of the innate immune system and play pivotal roles in host defense against pathogens and pathologic inflammatory reactions. ...

Real-Time Microscopy for Studying NETosis Kinetics and Screening Inhibitors

Real-Time, High-Throughput Microscopic Quantification of Human Neutrophil Extracellular Trap Release and Assessing the Pharmacology of Antagonists

Neutrophils play an important role in innate immune defense by using several strategies, including the release of neutrophil extracellular traps (NETs) in a process referred to as NETosis. However, in the past two decades, it has become clear that the accumulation of NETs in tissues contributes to the pathophysiology of multiple inflammatory and autoimmune diseases. Therefore, interest in the development of NETosis antagonists has risen. Variable and non-standardized methods to detect and analyze NETosis were developed concomitantly, each with its own advantages and limitations. Here, we describe a real-time microscopy method for the quantification of human NET release, allowing to study NETosis as well as NET inhibition in a high-throughput manner. The surface area-based semi-automated analysis recognizes NETs and distinguishes them from non-netting activated neutrophils. We demonstrate that the non-physiological NETosis inducers, calcium ionophore and phorbol-12-myristate-13-acetate (PMA), trigger the release of NETs with different characteristics and kinetics. Furthermore, we show that this approach allows studying NET release in response to disease-relevant stimuli, including immune complexes, N-Formylmethionine-leucyl-phenylalanine (fMLF), monosodium urate crystals, and calcium pyrophosphate crystals. To exemplify the utility of this method to study NETosis antagonists, we used CIT-013, a first-in-class monoclonal antibody inhibitor of NET release. CIT-013 targets citrullinated histone H2A and H4 and efficiently inhibits NET release with an IC50 of 4.6 nM. Other anti-histone antibodies tested lacked this NETosis-inhibitory capacity. Altogether, we demonstrate that this protocol enables specific, reliable, and reproducible high-throughput quantification of NETs, enhancing the study of NET release characteristics, kinetics, and pharmacology of NETosis antagonists.

This defined protocol describes a real-time high-throughput microscopy approach to visualize and quantify human neutrophil extracellular trap (NET) release in vitro. The reproducible method allows investigation of the characteristics and kinetics of NET release upon stimulation with distinct NETosis inducers and enables assessment of the pharmacology of NETosis antagonists.

Neutrophils play an important role in innate immune defense by using several strategies, including the release of neutrophil extracellular traps (NETs) in ...

A Degranulation Assay to Monitor Neutrophil Activation and Gelatinase Release

This video demonstrates an assay to monitor neutrophil activation and granule exocytosis through a degranulation assay. Upon activation by proinflammatory compounds, cytoplasmic granules within neutrophils containing gelatinase fuse with the cell membrane, discharging their cargo. The presence of gelatinase in the supernatant is assessed through the hydrolysis of a fluorophore-conjugated gelatin substrate, confirming neutrophil activation and degranulation.

The MUB₄₀ Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

Summary

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Chapters in this video

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Intravital Imaging of Neutrophil Priming Using IL-1β Promoter-driven DsRed Reporter Mice

Monitoring Neutrophil Elastase and Cathepsin G Activity in Human Sputum Samples

CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics

Quantifying Myeloperoxidase-DNA and Neutrophil Elastase-DNA Complexes from Neutrophil Extracellular Traps by Using a Modified Sandwich ELISA

A Set of Screening Techniques for a Quick Overview of the Neutrophil Function

Detecting Migration and Infiltration of Neutrophils in Mice

Real-Time, High-Throughput Microscopic Quantification of Human Neutrophil Extracellular Trap Release and Assessing the Pharmacology of Antagonists

A Degranulation Assay to Monitor Neutrophil Activation and Gelatinase Release