Name: human lung dendritic cells: spatial distribution and phenotypic identification in endobronchial biopsies using immunohistochemistry and flow cytometry
Uploaded: 2017-01-20T14:00:00
Description: Watch this Scientific Journal Video about Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry at JoVE

The authors would like to thank the volunteers who have contributed clinical material to this study. We are also thankful to the staff at the Department of Public Health and Clinical Medicine, Division of Medicine/Respiratory Medicine, University Hospital, Ume&#229; (Norrlands universitetssjukhus) for the collection of all clinical material.
This work was supported by grants to AS-S from the Swedish Research Council, the Swedish Heart-Lung Foundation, the Swedish Foundation for Strategic Research, and the Karolinska Institutet.

NOTE: This research was approved by the regional Ethical Review Board in Ume&#229;, Sweden.
1. Bronchoscopy for Sampling Endobronchial Biopsies from Human Subjects
<ol>
	<li>Obtain informed consent from all participants.</li>
	<li>Treat subjects with oral midazolam (4-8 mg) and intravenous glycopyrronium (0.2-04 mg) 30 min before the bronchoscopy. Apply topical anesthesia with lidocaine in the larynx and bronchi. Let the subject gargle with ~3 ml of lidocaine 4% and apply 3 ml to the tongue ...

<ol>
	<li>Kopf, M., Schneider, C., Nobs, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+and+function+of+lung-resident+macrophages+and+dendritic+cells.">The development and function of lung-resident macrophages and dendritic cells.</a> Nat Immunol. 16 (1), 36-44 (2015).</li><li>Condon, T. V., Sawyer, R. T., Fenton, M. J., Riches, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+dendritic+cells+at+the+innate-adaptive+immune+interface.">Lung dendritic cells at the innate-adaptive immune interface.</a> J Leukoc Biol. 90 (5), 883-895 (2011).</li><li>Lambrecht, B. N., Hammad, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+lung+dendritic+cells+at+the+origin+of+asthma.">Biology of lung dendritic cells at the origin of asthma.</a> Immunity. 31 (3), 412-424 (2009).</li><li>Schlitzer, A., McGovern, N., Ginhoux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+and+monocyte-derived+cells:+Two+complementary+and+integrated+functional+systems.">Dendritic cells and monocyte-derived cells: Two complementary and integrated functional systems.</a> Semin Cell Dev Biol. 41, 9-22 (2015).</li><li>Ziegler-Heitbrock, L., 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=Nomenclature+of+monocytes+and+dendritic+cells+in+blood.">Nomenclature of monocytes and dendritic cells in blood.</a> Blood. 116 (16), e74-e80 (2010).</li><li>Demedts, I. K., Brusselle, G. G., Vermaelen, K. Y., Pauwels, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+human+pulmonary+dendritic+cells.">Identification and characterization of human pulmonary dendritic cells.</a> Am J Respir Cell Mol Biol. 32 (3), 177-184 (2005).</li><li>Donnenberg, V. S., Donnenberg, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+rare-event+detection+and+analysis+of+dendritic+cell+subsets+in+broncho-alveolar+lavage+fluid+and+peripheral+blood+by+flow+cytometry.">Identification rare-event detection and analysis of dendritic cell subsets in broncho-alveolar lavage fluid and peripheral blood by flow cytometry.</a> Front Biosci. 8, s1175-s1180 (2003).</li><li>Masten, B. J., 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=Characterization+of+myeloid+and+plasmacytoid+dendritic+cells+in+human+lung.">Characterization of myeloid and plasmacytoid dendritic cells in human lung.</a> J Immunol. 177 (11), 7784-7793 (2006).</li><li>Ten Berge, B., 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=A+novel+method+for+isolating+dendritic+cells+from+human+bronchoalveolar+lavage+fluid.">A novel method for isolating dendritic cells from human bronchoalveolar lavage fluid.</a> J Immunol Methods. 351 (1-2), 13-23 (2009).</li><li>Yu, C. I., 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=Human+CD1c++dendritic+cells+drive+the+differentiation+of+CD103++CD8++mucosal+effector+T+cells+via+the+cytokine+TGF-beta.">Human CD1c+ dendritic cells drive the differentiation of CD103+ CD8+ mucosal effector T cells via the cytokine TGF-beta.</a> Immunity. 38 (4), 818-830 (2013).</li><li>Nicod, L. P., Lipscomb, M. F., Toews, G. B., Weissler, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+potent+and+poorly+functional+human+lung+accessory+cells+based+on+autofluorescence.">Separation of potent and poorly functional human lung accessory cells based on autofluorescence.</a> J Leukoc Biol. 45 (5), 458-465 (1989).</li><li>Sertl, K., 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=Dendritic+cells+with+antigen-presenting+capability+reside+in+airway+epithelium,+lung+parenchyma,+and+visceral+pleura.">Dendritic cells with antigen-presenting capability reside in airway epithelium, lung parenchyma, and visceral pleura.</a> J Exp Med. 163 (2), 436-451 (1986).</li><li>van Haarst, J. M., de Wit, H. J., Drexhage, H. A., Hoogsteden, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+and+immunophenotype+of+mononuclear+phagocytes+and+dendritic+cells+in+the+human+lung.">Distribution and immunophenotype of mononuclear phagocytes and dendritic cells in the human lung.</a> Am J Respir Cell Mol Biol. 10 (5), 487-492 (1994).</li><li>Schlitzer, 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=IRF4+transcription+factor-dependent+CD11b++dendritic+cells+in+human+and+mouse+control+mucosal+IL-17+cytokine+responses.">IRF4 transcription factor-dependent CD11b+ dendritic cells in human and mouse control mucosal IL-17 cytokine responses.</a> Immunity. 38 (5), 970-983 (2013).</li><li>Yu, Y. 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=Flow+Cytometric+Analysis+of+Myeloid+Cells+in+Human+Blood,+Bronchoalveolar+Lavage,+and+Lung+Tissues.">Flow Cytometric Analysis of Myeloid Cells in Human Blood, Bronchoalveolar Lavage, and Lung Tissues.</a> Am J Respir Cell Mol Biol. , (2015).</li><li>Haniffa, 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=Human+tissues+contain+CD141hi+cross-presenting+dendritic+cells+with+functional+homology+to+mouse+CD103++nonlymphoid+dendritic+cells.">Human tissues contain CD141hi cross-presenting dendritic cells with functional homology to mouse CD103+ nonlymphoid dendritic cells.</a> Immunity. 37 (1), 60-73 (2012).</li><li>Britten, K. M., Howarth, P. H., Roche, 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=Immunohistochemistry+on+resin+sections:+a+comparison+of+resin+embedding+techniques+for+small+mucosal+biopsies.">Immunohistochemistry on resin sections: a comparison of resin embedding techniques for small mucosal biopsies.</a> Biotech Histochem. 68 (5), 271-280 (1993).</li><li>Perfetto, S. P., Chattopadhyay, P. K., Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seventeen-colour+flow+cytometry:+unravelling+the+immune+system.">Seventeen-colour flow cytometry: unravelling the immune system.</a> Nat Rev Immunol. 4 (8), 648-655 (2004).</li><li>Baharom, 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=Dendritic+Cells+and+Monocytes+with+Distinct+Inflammatory+Responses+Reside+in+Lung+Mucosa+of+Healthy+Humans.">Dendritic Cells and Monocytes with Distinct Inflammatory Responses Reside in Lung Mucosa of Healthy Humans.</a> J Immunol. 196 (11), 4498-4509 (2016).</li><li>Salvi, S., 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=Acute+inflammatory+responses+in+the+airways+and+peripheral+blood+after+short-term+exposure+to+diesel+exhaust+in+healthy+human+volunteers.">Acute inflammatory responses in the airways and peripheral blood after short-term exposure to diesel exhaust in healthy human volunteers.</a> Am J Respir Crit Care Med. 159 (3), 702-709 (1999).</li><li>Schon-Hegrad, M. A., Oliver, J., McMenamin, P. G., Holt, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+density,+distribution,+and+surface+phenotype+of+intraepithelial+class+II+major+histocompatibility+complex+antigen+(Ia)-bearing+dendritic+cells+(DC)+in+the+conducting+airways.">Studies on the density, distribution, and surface phenotype of intraepithelial class II major histocompatibility complex antigen (Ia)-bearing dendritic cells (DC) in the conducting airways.</a> J Exp Med. 173 (6), 1345-1356 (1991).</li><li>Saeys, Y., Gassen, S. V., Lambrecht, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+flow+cytometry:+helping+to+make+sense+of+high-dimensional+immunology+data.">Computational flow cytometry: helping to make sense of high-dimensional immunology data.</a> Nat Rev Immunol. 16 (7), 449-462 (2016).</li></ol>

This paper describes how to generate a detailed spatial and phenotypical characterization of lung tissue-resident DCs in healthy humans using immunohistochemistry and flow cytometry on endobronchial mucosal biopsies collected during bronchoscopy. In the following paragraphs critical steps in the protocol are discussed in detail.
Critical Steps with the Protocol
Sectioning and immunohistochemistry: It is critical to keep the biopsy blocks at -20 &#176;C when not using t...

The lungs are in continuous contact with the external environment and are highly exposed to both harmless particles and microbes with the capacity to cause disease. Therefore, it is critical for the immune system to mount potent immune responses against invading pathogens, but it is equally important to maintain tolerance to inhaled antigens that do not cause disease. To provide potent immune surveillance, the respiratory system is lined with a network of immune cells, including dendritic cells (DCs). DCs are professional antigen-presenting cells with the unique capacity to activate naive T cells. In human lungs, resident DCs encounter an antigen and then process and transport it to the lung-draining lymph nodes for presentation to and activation of T cells1,2,3.
In the human immune system, DCs can be divided into several subsets, with distinct but overlapping functions: CD1c+ and CD141+ myeloid DCs (MDCs) and CD123+ plasmacytoid DCs (PDCs)4,5. While most detailed knowledge on human DCs stems from studies in blood, it is now evident that the human lungs also harbor rare populations of DC subsets with T-cell stimulatory capacity6,7,8,9. However, accumulating data show that immune cells, including DCs, differ in their frequency, phenotype, and function depending on their anatomical location10. Thus, it is important to study immune cells from the relevant tissue to understand their contribution to local immunity and tolerance. Taken together, this underlines the need to study lung-resident DCs when addressing lung diseases, despite blood DCs being more readily available and accessible in humans.
The first studies that investigated lung-resident DCs in humans primarily relied on morphology and the expression of single markers, such as HLA-DR and CD11c, in tissue sections using immunohistochemistry11,12,13. In contrast, more recent studies have typically relied on flow cytometric analyses to study different immune cell subsets. However, since it is difficult to find a single cell-surface marker that uniquely identifies a specific DC subset, the potential limitation of studies applying only four-color flow cytometry is the risk of including cell populations with similar phenotypic markers as DCs. For example, CD11c is expressed on all myeloid DCs and the vast majority of monocytes. On the other hand, in studies applying more advanced flow cytometry panels, non-cancerous lung tissue from surgical resections of patients were typically used10,14,15,16, although it is unclear whether these rare populations are truly representative of DCs present in healthy subjects. Overall, studies are limited largely due to the fact that surgically removed or whole human lung tissue is scarce.
To overcome some of these limitations, this work describes how to perform a detailed analysis of spatial distribution and a phenotypic identification of DCs in mucosal endobronchial biopsies obtained from healthy volunteers who undergo a bronchoscopy. Several small biopsies can be collected from each individual and can subsequently be embedded for sectioning and analysis using immunohistochemistry or enzymatically digested for advanced flow cytometric analysis. Using lung tissue in the form of endobronchial biopsies obtained from bronchoscopies confers the advantage of making it possible to perform the study on healthy volunteers, unlike open surgery of the lungs that, for obvious reasons, is limited to patients that require thoracic surgery. Furthermore, the tissue that is sampled during a bronchoscopy from healthy volunteers is physiologically normal, in contrast to a non-affected area of lung tissue in patients with a pulmonary disease. On the other hand, the biopsies are small and the number of cells retrieved, even when pooling several biopsies, limits the type of analyses that can be performed.
While the present work focuses on DCs, the methods described can be directly expanded to include other (immune) cells of interest that reside in human mucosal lung tissue. Furthermore, the protocols are also directly applicable to samples obtained from patients suffering from pulmonary diseases where bronchoscopy is part of establishing the diagnosis, such as chronic obstructive pulmonary disease (COPD), sarcoidosis, or lung cancer.

<table border="0" cellpadding="0" cellspacing="0" width="1260">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Bronchoscopy</td>
			<td style="width:172px;"></td>
			<td style="width:148px;"></td>
			<td style="width:628px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Bronchoscope BF1T160</td>
			<td style="width:172px;">Olympus</td>
			<td style="width:148px;">BF1T160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Light source&#160;</td>
			<td style="width:172px;">Olympus</td>
			<td style="width:148px;">Exera CV-160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Fenestrated forceps</td>
			<td style="width:172px;">Olympus</td>
			<td style="width:148px;">FB21C</td>
			<td>Used to take biopsies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Bite Block</td>
			<td style="width:172px;">Conmed</td>
			<td style="width:148px;">1429</td>
			<td>20x27mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Glucose 25%&#160;</td>
			<td style="width:172px;"></td>
			<td style="width:148px;"></td>
			<td>500mL intravenous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Glycopyrronium bromide 0.2mg/mL</td>
			<td style="width:172px;"></td>
			<td style="width:148px;"></td>
			<td>Intravenous. Prevents mucus/saliva secretion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Mixt. Midazolam 1mg/mL p.o</td>
			<td style="width:172px;"></td>
			<td style="width:148px;"></td>
			<td>Can be used for extra relaxation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Lidocaine, 40mg/mL</td>
			<td style="width:172px;"></td>
			<td style="width:148px;"></td>
			<td>Mouth and throat administration / Gargled</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Lidocaine 100mg/ml spray</td>
			<td style="width:172px;"></td>
			<td style="width:148px;"></td>
			<td>Administered to back of throat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Lidocaine 20mg/ml spray</td>
			<td style="width:172px;"></td>
			<td style="width:148px;"></td>
			<td>Administered via bronchoscope to airways</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Name of Reagent/ Equipment</td>
			<td style="width:172px;">Company</td>
			<td style="width:148px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">GMA processing and embedding</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Glass vials</td>
			<td style="width:172px;"></td>
			<td style="width:148px;"></td>
			<td>5mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Acetone</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:148px;">32201-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Molecular sieves, 4A</td>
			<td style="width:172px;">Alfa Aesar</td>
			<td style="width:148px;">88120</td>
			<td>3-4mm diameter pellets</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Phenylmethylsulfonyl fluoride</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:148px;">P-7626</td>
			<td>0.035g/100ml acetone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Iodoacetamide</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:148px;">I-6125</td>
			<td>0.37g/100ml acetone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Polythene-flat&#160; TAAB embedding capsules</td>
			<td style="width:172px;">TAAB laboratories</td>
			<td style="width:148px;">C094</td>
			<td>x500 8mm diameter, polythene, flat-bottom capsules</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Capsule holder</td>
			<td style="width:172px;">TAAB laboratories</td>
			<td style="width:148px;">C054</td>
			<td style="width:628px;">Holds 25 8mm capsules</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">JB-4 GMA embedding kit</td>
			<td style="width:172px;">Polysciences</td>
			<td style="width:148px;">00226</td>
			<td>Contains JB-4 Solution A (0026A-800), JB-4 solution B (0026B-3.8), benzoyl peroxide (02618-12)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Methyl benzoate</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:148px;">27614-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Silica gel with humidity indicator</td>
			<td style="width:172px;">Scharlau</td>
			<td style="width:148px;">GE0043</td>
			<td>2.5-6mm&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Name of Reagent/ Equipment</td>
			<td style="width:172px;">Company</td>
			<td style="width:148px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">GMA sectioning</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Glass microscope slides</td>
			<td style="width:172px;">ThermoFisher Scientific</td>
			<td style="width:148px;">10143562CEF</td>
			<td>Cut edges, frosted end</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Poly-L-Lysine solution</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:148px;">P8920-500mL</td>
			<td>1:10 for working solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sheet glass strips for ultramicrotomy</td>
			<td>Alkar</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P2287</td>
			<td>Wash solution (0.1% Tween20)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">LKB 7800B Knifemaker</td>
			<td>LKB</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Capsule splitter</td>
			<td style="width:172px;">TAAB laboratories</td>
			<td style="width:148px;">C065</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Carbon steel single edge blades</td>
			<td style="width:172px;">TAAB laboratories</td>
			<td style="width:148px;">B054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Vice</td>
			<td style="width:172px;"></td>
			<td style="width:148px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Ammonia, 25%</td>
			<td style="width:172px;">VWR</td>
			<td style="width:148px;">1133.1000</td>
			<td>2mL in 1L, 1:500 (0.05%)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Microtome</td>
			<td style="width:172px;">Leica</td>
			<td></td>
			<td>Leica RM 2165</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Light source</td>
			<td style="width:172px;">Leica</td>
			<td></td>
			<td>Leica CLS 150 XE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Microscope with swing arm stand</td>
			<td style="width:172px;">Leica</td>
			<td></td>
			<td>Leica MZ6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Name of Reagent/ Equipment</td>
			<td style="width:172px;">Company</td>
			<td style="width:148px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">GMA Immunohistochemistry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Diamond tipped pen</td>
			<td style="width:172px;">Histolab</td>
			<td style="width:148px;">5218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Hydrogen peroxide 30% solution</td>
			<td style="width:172px;">AnalaR Normapur</td>
			<td style="width:148px;">23619.264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S8032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris</td>
			<td>Roche</td>
			<td>10708976001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>VWR chemicals</td>
			<td>27810.295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin</td>
			<td>Millipore</td>
			<td>82-045-2</td>
			<td>Probumin BSA diagnostic grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s modified eagle medium (DMEM)</td>
			<td>Sigma-Aldrich</td>
			<td>D5546</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-human CD45 antibody</td>
			<td>BioLegend</td>
			<td>304002</td>
			<td>Mouse monoclonal, clone HI30, isotype IgG1k. Working concentration of 500 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-human CD1a antibody</td>
			<td>AbD Serotech</td>
			<td>MCA80GA</td>
			<td>Mouse monoclonal, clone NA1/34-HLK, isotype IgG2a. Working concentration of 10 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse monoclonal IgG1 isotype control</td>
			<td>Abcam</td>
			<td>ab27479</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse monoclonal IgG2a isotype control</td>
			<td>Dako</td>
			<td>X094301-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectastain ABC Elite standard kit</td>
			<td>Vector Labs</td>
			<td>PK-6100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:312px;">AEC (3-amino-9-ethylcarbazole) peroxidase substrate kite</td>
			<td style="width:172px;">Vector Labs</td>
			<td style="width:148px;">SK-4200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Mayers haematoxylin</td>
			<td style="width:172px;">HistoLab</td>
			<td style="width:148px;">01820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Permanent Aqueous Mounting Medium</td>
			<td style="width:172px;">AbD Serotech</td>
			<td style="width:148px;">BUF058C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Drying oven</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">DPX permanent mounting solution&#160;</td>
			<td style="width:172px;">VWR</td>
			<td style="width:148px;">360292F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Light microscope</td>
			<td style="width:172px;">Leica</td>
			<td></td>
			<td>Leica DMLB</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Microscope camera</td>
			<td style="width:172px;">Leica</td>
			<td></td>
			<td>Leica DFC 320</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Analysis software</td>
			<td style="width:172px;">Leica</td>
			<td></td>
			<td>Leica Qwin V3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Name of Reagent/ Equipment</td>
			<td style="width:172px;">Company</td>
			<td style="width:148px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Enzymatic digestion</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td>55021C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Dithiothreitol (DTT)</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td>DTT-RO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Collagenase II</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td>C6885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">DNase</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td>10104159001&#160;ROCHE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">RPMI 1640</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Forceps</td>
			<td style="width:172px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Platform rocker</td>
			<td style="width:172px;">Grant instruments</td>
			<td>PMR-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">50 mL conical tubes</td>
			<td style="width:172px;">Falcon</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">40 &#181;m cell strainer</td>
			<td style="width:172px;">Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Name of Reagent/ Equipment</td>
			<td style="width:172px;">Company</td>
			<td style="width:148px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Flow cytometry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Phosphate Buffered Saline (PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">LIVE/DEAD Aqua fixable dead cell stain kit</td>
			<td>Life Technologies</td>
			<td>L34957</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">CD45</td>
			<td>BD</td>
			<td>555485</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">CD3</td>
			<td>BD</td>
			<td>557757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">CD20</td>
			<td>BD</td>
			<td>335829</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">CD56</td>
			<td>Biolegend</td>
			<td>318332</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">CD66abce</td>
			<td>Miltenyi</td>
			<td>130-101-132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">HLA-DR</td>
			<td>BD</td>
			<td>555813</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">CD14</td>
			<td>BD</td>
			<td>557831</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">CD16</td>
			<td>Biolegend</td>
			<td>302026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">CD11c</td>
			<td>BD</td>
			<td>560369</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">CD1c</td>
			<td>Miltenyi</td>
			<td>130-098-009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">CD141</td>
			<td>Miltenyi</td>
			<td>130-090-514</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">CD103</td>
			<td>Biolegend</td>
			<td>350212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">LSR II Flow cytometer</td>
			<td>BD</td>
			<td></td>
			<td>Flow cytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">FlowJo</td>
			<td>FlowJo</td>
			<td></td>
			<td>Software for analysis</td>
		</tr>
	</tbody>
</table>

human lung dendritic cells: spatial distribution and phenotypic identification in endobronchial biopsies using immunohistochemistry and flow cytometry

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In order to maintain tolerance or to induce an immune response, the immune system must appropriately handle inhaled antigens. Lung dendritic cells (DCs) are essential in maintaining a delicate balance to initiate immunity when required without causing collateral damage to the lungs due to an exaggerated inflammatory response. While there is a detailed understanding of the phenotype and function of immune cells such as DCs in human blood, the knowledge of these cells in less accessible tissues, such as the lungs, is much more limited, since studies of human lung tissue samples, especially from healthy individuals, are scarce. This work presents a strategy to generate detailed spatial and phenotypic characterization of lung tissue resident DCs in healthy humans that undergo a bronchoscopy for the sampling of endobronchial biopsies. Several small biopsies can be collected from each individual and can be subsequently embedded for ultrafine sectioning or enzymatically digested for advanced flow cytometric analysis. The outlined protocols have been optimized to yield maximum information from small tissue samples that, under steady-state conditions, contain only a low frequency of DCs. While the present work focuses on DCs, the methods described can directly be expanded to include other (immune) cells of interest found in mucosal lung tissue. Furthermore, the protocols are also directly applicable to samples obtained from patients suffering from pulmonary diseases where bronchoscopy is part of establishing the diagnosis, such as chronic obstructive pulmonary disease (COPD), sarcoidosis, or lung cancer.

Studies characterizing human respiratory tissue-resident immune cells, including DCs, are limited, largely due to the fact that surgically removed or whole human lung tissue is scarce. Here, a less invasive method of obtaining lung tissue from endobronchial biopsies (EBB) of healthy volunteers and developed protocols to study the immune cells in the tissue using immunohistochemistry or flow cytometry are outlined.
Healthy volunt...

Watch this Scientific Journal Video about Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry at JoVE

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry

Lung-resident immune cells, including dendritic cells (DCs) in humans, are critical for defense against inhaled pathogens and allergens. However, due to the scarcity of human lung tissue, studies are limited. This work presents protocols to process human mucosal endobronchial biopsies for studying lung DCs using immunohistochemistry and flow cytometry.

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In ...

The main advantage of this technique is that a lot of information can be generated from small tissues obtained during bronchoscopy.This method can help answer key questions in mucosal immunology, where bronchoscopy is part of establishing the diagnosis, such as COPD, sarcoidosis, or lung cancer.Visual demonstration of this procedure will enable individuals new to this method to recognize when enzymatic digestion is complete.Demonstrating this procedure, along with Faezzah Baharom, will be Greg Rankin, a postdoc from our collaborator's lab at Ume

human-lung-dendritic-cells-spatial-distribution-phenotypic

Research

JoVE Journal

Immunology and Infection

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...