This work was funded by grants from Monash University, the National Health and Medical Research Council, and the Monash Lung and Sleep Institute. The authors would like to thank the staff of Clinical Immunology at Monash Health, Judy Callaghan, Alex Fulcher, Kirstin Elgass, and Camden Lo of Monash Micro Imaging (MMI) for help with the confocal microscopy imaging, and the authors acknowledge the MMI facilities of Monash University. The authors acknowledge the facilities, and scientific and technical assistance of the Monash Histology Platform.

This protocol follows experiments approved in: (1) humans by the Ethics Committee of Monash Medical Centre, and (2) animals by the Ethics Committee of the University of Melbourne.
1. Bronchoalveolar Lavage (BAL) Macrophages
<ol>
	<li>Use BAL to obtain lung macrophages in: (1) human subjects by bronchoscopy6, and (2) mice by using intra-tracheal aspiration13. 
	NOTE: Acquisition of the macrophages generally causes some activation of these c...

<ol>
	<li>Brinkmann, V., 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=Neutrophil+extracellular+traps+kill+bacteria.">Neutrophil extracellular traps kill bacteria.</a> Science. 303 (5663), 1532-1535 (2004).</li><li>Brinkmann, V., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps:+is+immunity+the+second+function+of+chromatin?.">Neutrophil extracellular traps: is immunity the second function of chromatin?.</a> J Cell Biol. 198 (5), 773-783 (2012).</li><li>Jorch, S. K., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+emerging+role+for+neutrophil+extracellular+traps+in+noninfectious+disease.">An emerging role for neutrophil extracellular traps in noninfectious disease.</a> Nat Med. 23 (3), 279-287 (2017).</li><li>Cheng, O. Z., Palaniyar, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NET+balancing:+a+problem+in+inflammatory+lung+diseases.">NET balancing: a problem in inflammatory lung diseases.</a> Frontiers immunol. 4, 1 (2013).</li><li>Boe, D. M., Curtis, B. J., Chen, M. M., Ippolito, J. A., Kovacs, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+traps+and+macrophages:+new+roles+for+the+versatile+phagocyte.">Extracellular traps and macrophages: new roles for the versatile phagocyte.</a> J Leukoc Biol. 97 (6), 1023-1035 (2015).</li><li>King, P. 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=Nontypeable+Haemophilus+influenzae+induces+sustained+lung+oxidative+stress+and+protease+expression.">Nontypeable Haemophilus influenzae induces sustained lung oxidative stress and protease expression.</a> PloS one. 10 (3), e0120371 (2015).</li><li>O'Sullivan, K. M., 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=Renal+participation+of+myeloperoxidase+in+antineutrophil+cytoplasmic+antibody+(ANCA)-associated+glomerulonephritis.">Renal participation of myeloperoxidase in antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis.</a> Kidney Int. 88 (5), 1030-1046 (2015).</li><li>Fuchs, T. 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=Novel+cell+death+program+leads+to+neutrophil+extracellular+traps.">Novel cell death program leads to neutrophil extracellular traps.</a> J Cell Biol. 176 (2), 231-241 (2007).</li><li>Wang, Y., 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=Histone+hypercitrullination+mediates+chromatin+decondensation+and+neutrophil+extracellular+trap+formation.">Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation.</a> J Cell Biol. 184 (2), 205-213 (2009).</li><li>Papayannopoulos, V., Metzler, K. D., Hakkim, A., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+elastase+and+myeloperoxidase+regulate+the+formation+of+neutrophil+extracellular+traps.">Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps.</a> J Cell Biol. 191 (3), 677-691 (2010).</li><li>Rohrbach, A. S., Slade, D. J., Thompson, P. R., Mowen, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+PAD4+in+NET+formation.">Activation of PAD4 in NET formation.</a> Front Immunol. 3, 360 (2012).</li><li>Lewis, H. D., 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=Inhibition+of+PAD4+activity+is+sufficient+to+disrupt+mouse+and+human+NET+formation.">Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation.</a> Nat Chem Biol. 11 (3), 189-191 (2015).</li><li>Ruwanpura, S. M., McLeod, L., Dousha, L. 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=Therapeutic+Targeting+of+the+IL-6+Trans-Signaling/Mechanistic+Target+of+Rapamycin+Complex+1+Axis+in+Pulmonary+Emphysema.">Therapeutic Targeting of the IL-6 Trans-Signaling/Mechanistic Target of Rapamycin Complex 1 Axis in Pulmonary Emphysema.</a> Am J Respir Crit Care Med. 194 (12), 1494-1505 (2016).</li><li>Brinkmann, V., Laube, B., Abu Abed, U., Goosmann, C., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps:+how+to+generate+and+visualize+them.">Neutrophil extracellular traps: how to generate and visualize them.</a> Journal of visualized experiments : JoVE. (36), (2010).</li><li>Chow, O. A., von Kockritz-Blickwede, M., Bright, A. 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=Statins+enhance+formation+of+phagocyte+extracellular+traps.">Statins enhance formation of phagocyte extracellular traps.</a> Cell host &amp; microbe. 8 (5), 445-454 (2010).</li><li>Wong, K. W., Jacobs, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterium+tuberculosis+exploits+human+interferon+gamma+to+stimulate+macrophage+extracellular+trap+formation+and+necrosis.">Mycobacterium tuberculosis exploits human interferon gamma to stimulate macrophage extracellular trap formation and necrosis.</a> J Infect Dis. 208 (1), 109-119 (2013).</li></ol>

The method described in this review is based on that used for defining the presence of NETs14. Macrophages are by far the dominant cell type in BAL samples and this method of collection is particularly suited for studying METs. If red blood cells are present in the BAL, these cells should be lysed by using ammonium chloride. The BAL procedure will generally activate the macrophages and therefore it is expected that there will be METs present in un-stimulated samples. The BAL macrophages are usuall...

Neutrophil extracellular traps (NETs), were first described by Brinkmann et al.1 They are predominantly produced in response to infection (especially to bacteria) and have an important role in host defense1,2. They have also been described to occur in response to non-infectious disease, including vasculitis and systemic lupus erythematosus (SLE); and to the mitogen phorbol 12-myrisate 13-acetate (PMA)2,3. It has recently been recognized that other cell types may also produce extracellular traps, including macrophages. Macrophage extracellular traps (METs) are not yet a well-defined entity in the literature4,5. We have recently established methods to detect the presence of METs both in vitro and in vivo6,7. In this article, the measurement of METs using confocal microscopy will be described.
Key features of NETosis which distinguish it from other cellular pathways (such as apoptosis8) are the extrusion of chromatin in conjunction with: (1) citrullination of histones (H3Cit)9, (2) co-expression of granule proteases10, and (3) involvement of peptidyl arginine deiminase (PAD) 411,12. Macrophages also express H3Cit, granule proteases, and PAD, and these features can be used to define the presence of METs.
METs may have a particularly important role in the lung, as the macrophage is the dominant cell present in the alveoli and airways of the lung and has the initial role in directing the cellular immune response to infection/inflammation. In addition, while much of the lung is empty space (e.g., within the alveoli), the METs are potentially able to expand into the space available, in contrast to solid organs.
The most widely used method to define the presence of NETs is by confocal microscopy. There is not yet a clearly defined way to measure METs. The technique for the measurement of NETs has been adapted to measure the presence of METs in this protocol. The main requirements for this method are access to confocal microscopy and appropriate imaging software for analysis.

<table>
	<tbody>
		<tr>
			<td>Human samples: Primary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-human H3Cit (Citrulline R26)</td>
			<td>Abcam</td>
			<td>AB5103</td>
			<td>10 ug/ml</td>
		</tr>
		<tr>
			<td>Rabbit anti-human MMP12</td>
			<td>Novus Biological</td>
			<td>NB110-57214</td>
			<td>1:100 concentration not quantifiable</td>
		</tr>
		<tr>
			<td>Mouse anti-human MMP9</td>
			<td>Novus Biological</td>
			<td>AB119906</td>
			<td>1:200 ascites, no concentration given</td>
		</tr>
		<tr>
			<td>Rabbit anti-human PADI2/PAD2</td>
			<td>Abcam</td>
			<td>AB16478</td>
			<td>7 ug/ml</td>
		</tr>
		<tr>
			<td>Mouse anti-human PADI4/PAD4</td>
			<td>Abcam</td>
			<td>AB128086</td>
			<td>10.1 ug/ml</td>
		</tr>
		<tr>
			<td>Sheep anti-human NE</td>
			<td>LifeSpan Bioscience</td>
			<td>LS-B4244</td>
			<td>25 ug/ml</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Mouse samples: Primary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse MMP9</td>
			<td>Abcam</td>
			<td>AF909</td>
			<td>10 ug/ml</td>
		</tr>
		<tr>
			<td>Rabbit anti-mouse H3Cit (Citrulline R26)</td>
			<td>Abcam</td>
			<td>AB5103</td>
			<td>10 ug/ml</td>
		</tr>
		<tr>
			<td>Rat anti-mouse F4/80</td>
			<td>In-house (hybridoma)</td>
			<td>In house</td>
			<td>20 ug/ml</td>
		</tr>
		<tr>
			<td>Sheep anti-human Anti-HNE /NE</td>
			<td>Sapphire Bioscience</td>
			<td>LS-B4244</td>
			<td>25 ug/ml</td>
		</tr>
		<tr>
			<td>Mouse anti-human &#173;&#173;&#173;&#173;PADI4/PAD4</td>
			<td>Abcam</td>
			<td>AB128086</td>
			<td>10.1 ug/ml</td>
		</tr>
		<tr>
			<td>Super-frost plus slides</td>
			<td>Menzel</td>
			<td>S41104A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dapi-prolong gold</td>
			<td>Molecular probes</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>Sigma-Aldrich</td>
			<td>85111</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>E9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken anti-rabbit AF 488/</td>
			<td>Life technologies</td>
			<td>A-21441</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken anti-rabbit AF 594</td>
			<td>Life technologies</td>
			<td>A-21442</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken anti-goat AF 594</td>
			<td>Life technologies</td>
			<td>a-21468</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken anti-mouse AF488</td>
			<td>Life technologies</td>
			<td>A-21200</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-sheep AF 594</td>
			<td>Life technologies</td>
			<td>A-11018</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken anti-mouse AF 647</td>
			<td>Life technologies</td>
			<td>A-21463</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-sheep AF 594</td>
			<td>Life technologies</td>
			<td>A-11016</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotype control</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit IgG</td>
			<td>In house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit IgG</td>
			<td>In house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG1</td>
			<td>BD Bioscience</td>
			<td>550878</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit IgG</td>
			<td>In house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG2a</td>
			<td>BioLegend</td>
			<td>400201</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheep IgG</td>
			<td>In house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software Programs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>To average intensity of fluorphores a standard office application like Microsoft Excel can be used</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Microscopes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C1 Confocal scanning microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FV1200 Confocal scanning microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Tissue sources</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human BAL samples from the bronchoscopy suite at Monash Medical Centre</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse BAL samples and lung tissue from the Department of Pharmacology, University of Melbourne.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Sigma-Aldrich</td>
			<td>r8758</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Sigma-Aldrich</td>
			<td>F0926</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Other reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sudan black</td>
			<td>Sigma-Aldrich</td>
			<td>199664</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde/periodate/lysine (PLP) fixative</td>
			<td>Sigma-Aldrich</td>
			<td>27387</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sigma-Aldrich</td>
			<td>214736</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Sigma-Aldrich</td>
			<td>K2753</td>
			<td></td>
		</tr>
		<tr>
			<td>Natural formalin</td>
			<td>Sigma-Aldrich</td>
			<td>42904</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Sigma-Aldrich</td>
			<td>327204</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A2576</td>
			<td></td>
		</tr>
		<tr>
			<td>Solvent 3B</td>
			<td>Hi-Chem</td>
			<td>2026</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips 12 ml</td>
			<td>Sigma-Aldrich</td>
			<td>S1815</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips 60 x 24 ml</td>
			<td>Sigma-Aldrich</td>
			<td>C6875</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>c57 black 6</td>
			<td>Monash animal research platform (MARP)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BALB/c</td>
			<td>MARP</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing macrophage extracellular traps using confocal microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

METs may be visualized from BAL samples, in lung tissue and in thicker lung sections with 3-D images. An example of METs visualized in a BAL sample is shown in Figure 1. The morphology of the METs will vary according to their stage of maturation. The first detectable feature on microscopy is the movement of the nucleus to the edge of the cell. This is followed by extracellular chromatin with other co-expressed mediators, such as H3Cit and granule proteases. I...

Watch this Scientific Journal Video about Visualizing Macrophage Extracellular Traps Using Confocal Microscopy at JoVE.com

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

The overall goal of this method is to demonstrate how macrophage extracellular traps can be visualized and studied using confocal microscopy. This method can help answer key questions in the inflammation field, such as responses to infection and other immune stimuli. The main advantage of this technique is that it is a modification of a well established method that has been used to study neutrophil extracellular traps.Demonstrating this procedure will be Roleen Sharma. After obtaining lung macrophages from humans and mice by bronchoalveolar lavage, or BAL, according to the text protocol, use trypan blue and a hemocytometer to perform a viable cell count on the BAL solution. Pipette one to four times 10 to the fifth macrophages in 500 microliters of culture medium onto cover slips in the wells of 24-well plates, then incubate the cells at 37 degrees Celsius overnight for the cells to adhere.Remove the culture medium and use PBS to wash the cells once. Then add 2%paraformaldehyde-periodate-lysine, or PLP fixative, and incubate the samples for 10 minutes. After using PBS to briefly wash the cells, add 0.2%TWEEN 20 in PBS and incubate the cells for 20 minutes.Next, to block the cells, add 10%chicken serum in 5%BSA, diluted in PBS and incubate the samples at room temperature for 30 minutes. Then replace the block solution with a one to 100 concentration of primary and isotope control antibodies and incubate the cells at room temperature for one hour. Following the incubation, use PBS to wash the cells before adding the corresponding secondary antibodies.Then incubate the samples for 40 minutes at room temperature. After washing the cells in PBS, mount the samples with DAPI based mounting medium, then visualize the samples using a confocal microscope. To pretreat the FFPE samples with antigen retrieval solution, oven dry the slides at 60 degrees Celsius for 60 minutes.Then transfer the slides into xylene solution for 30 minutes before transferring the samples to 70%ethanol at room temperature for five minutes. After using tap water to rinse the slides, place them in heat-proof plastic wrap and subject them to higher antigen retrieval by placing them in a pressure cooker in Tris-EDTA pH 9.0 for 10 minutes. Cool the samples for 20 minutes and transfer them to tap water.Then place them on a rocker for five minutes. Repeat the tap water wash and then wash the slides once in PBS on the rocker. To block the samples, add 10%chicken serum in 5%BSA PBS and incubate them at room temperature for 30 minutes.Then, to define macrophage extracellular traps, or METs in macrophages, add a one to 100 dilution of primary antibodies in 1%BSA PBS and incubate the slides at four degrees Celsius for 16 hours. Use PBS to wash the samples two times on a rocker for five minutes each. Add the corresponding fluorescent secondary antibodies in 1%BSA PBS and incubate the slides at room temperature for 40 minutes.Following PBS washes, use DAPI containing mounting medium to stain the chromatin and mount the samples. Using a confocal laser scanning head attached to an inverted microscope, capture fluorescent images using 20X 0.1 NA air and 40X 1.0 NA oil objectives. Capture single plane 512 by 512 pixel images by clicking on the Line Sequential leveling button.Obtain at least 10 fields of view per section for analysis and data for each result. To stain the sections in microcentrifuge tubes, add the relevant primary antibodies in 200 microliter volumes and incubate the samples at four degrees Celsius for 16 hours. Wash the sections in PBS three times for 10 minutes before adding the appropriate secondary antibodies and incubating the samples at room temperature for one hour.Use DAPI to stain the lung sections in solution and incubate the samples for 20 minutes before adding cover slips to the sections on microscope slides in PBS. Use an inverted confocal microscope with a 40X 1.3 NA objective, equipped with 405 nanometers, 440 nanometers, 473 nanometers, 543 nanometers, and 635 nanometer lasers, to capture images and to record 3D Z stacks, 0.54 microns thick, with optimal Z sectioning. Finally, analyze the images according to the text protocol.An example of METs in a BAL sample is shown here. The first detectable feature is the movement of the nucleus to the edge of the cell, as seen in this earlier stage MET that has a roughly spherical shape. This is followed by extracellular chromatin with other co-expressed mediators, such as H3Cit and granular proteases, as seen in this more mature MET.Earlier stage MET is upper left and later stage MET is below this towards the center. Lung tissue METs are shown in this figure. As the lungs are inflated with fluid to define the lung architecture, the METs will be pushed against the alveolar walls.The morphology of the METs will also vary depending on in which plane the tissue was cut. The standard sections used for lung tissue samples are four to five microns thick. Thicker tissue sections enable multiple images to be taken and the METs can then be viewed in 3D.An example of a 3D image from mouse lung tissue cut in 25 to 35 micron sections is presented here. Once mastered, this technique can be done in two days of staining plus imaging if it's performed properly. While attempting this procedure, it is important to remember to be gentle with the washes.Following this procedure, other methods like zymography can be performed to answer additional questions like protease expression. After its development, this technique may pave way for the study of inflammatory macrophage responses. After watching this video, you should have a good understanding of how to visualize METs using confocal microscopy.Don't forget that working with PLP can be hazardous and precautions such as wearing gloves and eye protection should always be taken while performing this procedure.

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Research

JoVE Journal

Immunology and Infection

10.0K vistas.  Monash Medical Centre. The overall goal of this method is to demonstrate how macrophage extracellular traps can be visualized and studied using confocal microscopy. This method can help answer key questions in the inflammation field, such as responses to infection and other immune stimuli. The main advantage of this technique is that it is a modification of a well established method that has been used to study neutrophil extracellular traps.Demonstrating this procedure will be Roleen Sharma. After obtaining ...