Name: visualizing macrophage extracellular traps using confocal microscopy
Uploaded: 2017-10-19T14:00:00
Description: Watch this Scientific Journal Video about Visualizing Macrophage Extracellular Traps Using Confocal Microscopy at JoVE.com

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.

visualizing-macrophage-extracellular-traps-using-confocal-microscopy

Research

JoVE Journal

Immunology and Infection

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

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

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular interaction that is not clearly understood is non-lytic exocytosis. In this process, yeast cells are released into the extracellular space by a poorly understood mechanism that leaves both the macrophage and Cn viable. Here, we describe how to follow a large number of individually infected macrophages for a 24 hr infection period by time-lapsed microscopy. Infected macrophages are housed in a heating chamber with a CO2 atmosphere attached to a microscope that provides the same conditions as a cell-culture incubator. Live digital microscopy can provide information about the dynamic interactions between a host and pathogen that is not available from static images. Being able to visualize each infected cell can provide clues as to how macrophages handle fungal infections, and vice versa. This technique is a powerful tool in studying the dynamics that are behind a complex phenomenon.

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

We describe how to visualize macrophage-C. neoformans (Cn) interactions in real time, with specific emphasis on the process of non-lytic exocytosis using digital light microscopy. Using this technique individually infected macrophages can be studied to ascertain various aspects of this phenomenon.

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular ...

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions.
In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves.
A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained.
We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

In the following protocol, we describe a very simple way to isolate Neutrophil Extracellular traps (NETs) from human whole blood using readily available reagents. We then demonstrate how the isolated NETs can be used in an in vitro adhesion assay with cancer cells.

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role ...

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. Immunofluorescence microscopy and image-based quantification methods remain important tools to quantitate neutrophil extracellular trap formation. However, there are key limitations to the immunofluorescence-based methods that are currently available for quantifying NETs. Manual methods of image-based NET quantification are often subjective, prone to error and tedious for users, especially non-experienced users. Also, presently available software options for quantification are either semi-automatic or require training prior to operation. Here, we demonstrate the implementation of an automated immunofluorescence-based image quantification method to evaluate NET formation called NETQUANT. The software is easy to use and has a user-friendly graphical user interface (GUI). It considers biologically relevant parameters such as an increase in the surface area and DNA:NET marker protein ratio, and nuclear deformation to define NET formation. Furthermore, this tool is built as a freely available app, and allows for single-cell resolution quantification and analysis.

Here, we present a protocol for generating neutrophil extracellular traps (NETs) and operating NETQUANT, a fully automatic software option for quantification of NETs in immunofluorescence images.

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. ...

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, periodontitis, mucosal infections and, most notably, early childhood caries. In all of these conditions, the pH in the biofilm matrix impacts microbe-host interactions and thus the disease progression. The present protocol describes a confocal microscopy-based method to monitor pH dynamics inside cross-kingdom biofilms comprising Candida albicans and Streptococcus mutans. The pH-dependent dual-emission spectrum and the staining properties of the ratiometric probe C-SNARF-4 are exploited to determine drops in pH in extracellular areas of the biofilms. Use of pH ratiometry with the probe requires a meticulous choice of imaging parameters, a thorough calibration of the dye, and careful, threshold-based post-processing of the image data. When used correctly, the technique allows for the rapid assessment of extracellular pH in different areas of a biofilm and thus the monitoring of both horizontal and vertical pH gradients over time. While the use of confocal microscopy limits Z-profiling to thin biofilms of 75 &#181;m or less, the use of pH ratiometry is ideally suited for the noninvasive study of an important virulence factor in cross-kingdom biofilms.

The protocol describes the cultivation of cross-kingdom biofilms consisting of Candida albicans and Streptococcus mutans and presents a confocal microscopy-based method for the monitoring of extracellular pH inside these biofilms.

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, ...

Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy

Neutrophil extracellular traps (NETs), composed of cell-free DNA (cfDNA) and proteins like histones and neutrophil elastase (NE), are released by neutrophils in response to systemic inflammation or pathogens. Although NETs have previously been shown to augment clot formation and inhibit fibrinolysis in humans and dogs, the role of NETs in cats with cardiogenic arterial thromboembolism (CATE), a life-threatening complication secondary to hypertrophic cardiomyopathy, is unknown. A standardized method to identify and quantify NETs in cardiogenic arterial thrombi in cats will advance our understanding of their pathological role in CATE. Here, we describe a technique to identify NETs in formaldehyde-fixed and paraffin-embedded thrombi within the aortic bifurcation, extracted during necropsy. Following deparaffinization with xylene, aortic sections underwent indirect heat-induced antigen retrieval. Sections were then blocked, permeabilized, and ex vivo NETs were identified by colocalization of cell-free DNA (cfDNA), citrullinated histone H3 (citH3), and neutrophil elastase (NE) using immunofluorescence microscopy. To optimize the immunodetection of NETs in thrombi, autofluorescence of tissue elements was limited by using an autofluorescence quenching process prior to microscopy. This technique could be a useful tool to study NETs and thrombosis in other species and offers new insights into the pathophysiology of this complex condition.

We describe a method to identify neutrophil extracellular traps (NETs) in formaldehyde-fixed and paraffin-embedded feline cardiogenic arterial thrombi using heat-induced antigen retrieval and a double immunolabeling protocol.

Neutrophil extracellular traps (NETs), composed of cell-free DNA (cfDNA) and proteins like histones and neutrophil elastase (NE), are released by ...

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3).
Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

The mechanism associated with phagocytosis in Leishmania infection remains poorly understood. Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells.

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up ...

Morphological and Compositional Analysis of Neutrophil Extracellular Traps Induced by Microbial and Chemical Stimuli

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of neutrophil extracellular traps (NETs). This study analyzes the morphological and compositional changes in NETs induced by microbial and chemical stimuli using standardized in vitro methodologies for NET induction and characterization with human cells. The procedures described here allow the analysis of NET morphology (lytic or non-lytic) and composition (DNA-protein structures and enzymatic activity), and the effect of soluble factors or cellular contact on such characteristics. Additionally, the techniques described here could be modified to evaluate the effect of exogenous soluble factors or cellular contact on NET composition.
The applied techniques include the purification of polymorphonuclear cells from human peripheral blood using a double density gradient (1.079-1.098 g/mL), guaranteeing optimal purity and viability (&#8805; 95%) as demonstrated by Wright's staining, trypan blue exclusion, and flow cytometry, including FSC versus SSC analysis and 7AAD staining. NET formation is induced with microbial (Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans) and chemical (phorbol myristate acetate, HOCl) stimuli, and the NETs are characterized by DNA-DAPI staining, immunostaining for the antimicrobial peptide cathelicidin (LL37), and quantification of enzymatic activity (neutrophil elastase, cathepsin G, and myeloperoxidase). The images are acquired through fluorescence microscopy and analyzed with ImageJ.

Presented here is a protocol for the induction and analysis of in vitro neutrophil extracellular traps (NETs). Quantification of DNA, cathelicidin (LL37), and enzyme activity yielded data that show the variability in the composition and morphology of NETs induced by microbial and chemical stimuli under similar controlled conditions.