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 th...

<ol>
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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. 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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 border="0" cellpadding="0" cellspacing="0" style="width:895px;" width="894">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">MUB40-Cy5</td>
			<td style="width:123px;">Benoit Marteyn</td>
			<td style="width:344px;">benoit.marteyn@pasteur.fr</td>
			<td style="width:167px;">Fluorescent MUB40 peptide (available with other conjugated fluorophores)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">RI-MUB40-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 height="40">
			<td height="40" style="height:40px;">Parformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td align="right">16005</td>
			<td>Fixative for histology</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S7903</td>
			<td>Solution used to remove excess PFA</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Optimal Cutting Temperature Compound (OCT)</td>
			<td>Sakura Finetek USA</td>
			<td align="right">4583</td>
			<td>Used to freeze tissue before cryostat sectioning</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">2-Methylbutane</td>
			<td>Sigma-Aldrich</td>
			<td>M32631</td>
			<td>Used to freeze tissue before cryostat sectioning</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td align="right">1.00974</td>
			<td>Used for dry ice bath to freeze tissue</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Leica Cryostat</td>
			<td>Leica Biosystems</td>
			<td>CM1520</td>
			<td>Used to section tissues</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass microscopy slides</td>
			<td>Fisher Scientific</td>
			<td>12-518-101</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cover slips</td>
			<td>Thor Labs</td>
			<td>CG15CH</td>
			<td>Hi precision coverslip</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dako pen&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>Z377821</td>
			<td>Used to prevent liquid loss during tissue staining</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Saponin</td>
			<td>Sigma-Aldrich</td>
			<td align="right">47036</td>
			<td>Used to permeabilize cells for IF staining</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DAPI</td>
			<td>Sigma-Aldrich</td>
			<td align="right">10236276001</td>
			<td>Stains DNA&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Alexa fluor 488 Phalloidin&#160;</td>
			<td>Fisher Scientific</td>
			<td>A12379</td>
			<td>Binds actin</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Prolong Gold antifade Mountant</td>
			<td>Fisher Scientific</td>
			<td>P36930</td>
			<td>Prolongs IF signal and resistance to photobleaching</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fluorescent microscope</td>
			<td>Various</td>
			<td>Various</td>
			<td>Used to image IF slides</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anoxic Cabinet</td>
			<td>Various</td>
			<td>Various</td>
			<td>Used for the purification of live inactivated neutrophils</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium Chloride NaCL</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E9884</td>
			<td>Washing buffer component</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MACS BSA buffer</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td>Washing buffer component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Percoll</td>
			<td>Sigma-Aldrich</td>
			<td>P4937</td>
			<td>Gradient for neutrophil purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD235a glycophorin magnetic microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-050-501</td>
			<td>Used to remove contaminating RBCs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td>Used in the removal of RBCs from neutrophils</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 ...

MUB40 is a novel neutrophil marker, which binds to lactoferrin and allows a specific detection in human and all mammal tissue sample tested so far. Labeling neutrophils with MUB40 is easy, quick, and specific. It's now used widely in our lab and the implication of this technique extend towards diagnosis of inflammatory diseases.To begin, prepare solutions one, two, and three according to the text protocol, and place them in an anoxic cabinet overnight. Then, centrifuge the tubes of collected blood at 650 g for 20 minutes to separate the cells from the plasma fraction. Without disturbing the separated blood, transfer the tubes to the anoxic cabinet and transfer the plasma fractions to a 50 milliliter conical tube.After this remove the tube of plasma from the anoxic cabinet and centrifuge it at 2900 g for 20 minutes. Taking care not to disrupt the pelleted platelets, transfer the tube to the anoxic cabinet and pipette the platelet poor plasma into a fresh 50 milliliter tube. Using the blood collection tubes, combine the red blood cells in a conical 50 milliliter tube.Then, add 0.9%sodium chloride solution until the total volume reaches 44 milliliters, and add six milliliters of 6%dextran. Invert the tube 10 to 20 times to gently mix the blood and dextran mixture, and allow the tube to sediment for at least 30 minutes. While the tube sediments, prepare a Percoll gradient by adding 4.2 milliliters of Percoll solution to 5.8 milliliters of plasma, and invert the tube to mix.Next, collect the neutrophil containing top fraction of the dextran, taking care not to pipette red blood cells. Tighten the screw cap, remove the dextran tube from the anoxic cabinet, and centrifuge the tube at 300 g for 10 minutes. Taking care not to disturbed the pelleted cells, place the tube back in the anoxic cabinet.Then, remove the liquid by pipetting. Gently resuspend the pellet in one milliliter of plasma. Slowly add the resuspended cells to the tops of the Percoll solution.Carefully remove the Percoll solution tube from the anoxic cabinet and centrifuge it at 800 g for 20 minutes to separate PBMCs from neutrophils. Next, prepare 14 milliliters of washing buffer solution according to the text protocol. Then, place the Percoll tube in the anoxic cabinet.Use a pipette to remove the Percoll and PBMCs and resuspend the neutrophil containing pellet in one milliliter of washing buffer solution. Next, add 200 microliters of magnetic beads to the resuspended cells. Gently mix and incubate the cells and beads for at least 15 minutes.After this, wash a separation column twice, with two milliliters of washing buffer. While holding a new, 15 milliliter conical tube under the column, slowly pipette the neutrophil red blood cell mixture through the column. The flow-through from the column should be cloudy and lose its red color, confirming the total separation of neutrophils from remaining red blood cells.Add two milliliters of washing buffer to the top of the column and collect the flow-through in the same tube. By the end of this wash, the flow-through should be clear. Gently mix the collection tube to ensure the neutrophils are evenly distributed.Then collected 10 microliters of the flow-through from cell counting. After this, remove the neutrophil tube from the anoxic cabinet and centrifuge the tube at 300 g for 20 minutes. Place the sedimented neutrophils back into the anoxic cabinet and remove the washing buffer via pipetting.Then, resuspend the pellet in plasma. Add one microliter of RI-MUB40-Cy5 to one milliliter of RPMI-1640 medium, without phenol red, and mix via pipetting. Then, add one microliter of FMLP solution and pipette to mix.Transfer the mixture to a glass bottom microscopy dish. Then add an appropriate volume of purified cells to the dish. Finally, place the dish into an inverted fluorescent microscope and start image acquisition.In this protocol, MUB40 was used on living cells to detect neutrophil secretion upon simulation with FMLP. The dynamics of the release of granule content, including lactoferrin, may be visualized over time, here in magenta. MUB40 may be additionally used on fixed neutrophils.Here, phagocytizing fluorescent beads in a timed series. Neutrophil granule's content is labeled here in blue. The neutrophils showed little to no cell staining at early time points and gradually, RI-MUB40 labeling increased over time, both on the plasma membrane and bead surface.MUB40 may also be used on fixed inflammatory tissues to detect neutrophils specifically, as shown in the text figures. We describe here the neutrophil purification procedure, applied routinely in our lab. Any other method is suitable, MUB40-Cy5 labeling will still work.Neutrophil may be specifically labeled with MUB40 after purification, or directly in inflamed tissue from patients or animal models. Several MUB40 derivatives have been designed to broaden its use. After its development, this technique paved the way for researcher in the field of inflammation, to explore neutrophilic recruitment and activation in inflammatory diseases.

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6.0K Views.  Institut Pasteur. MUB40 è un nuovo marcatore neutrofilo, che si lega alla lattoferrina e consente un rilevamento specifico nel campione di tessuto umano e di tutti i mammiferi testato finora. Etichettare i neutrofili con MUB40 è facile, veloce e specifico. Ora è ampiamente usato nel nostro laboratorio e l'implicazione di questa tecnica si estende verso la diagnosi di malattie infiammatorie.Per iniziare, preparare le soluzioni una, due e tre in base al protocollo di testo e posizionarle in un armadio ...