JSO is supported by an NIH Ruth L. Kirschstein NRSA (F32 DK091040), AK is supported by a Postdoctoral Fellowship from the American Diabetes Association (7-10-MI-05), and AHH is supported by an American Heart Association Established Investigator Award (12EIA8270000). Flow cytometry experiments were performed in the VMC Flow Cytometry Shared Resource. The VMC Flow Cytometry Shared Resource is supported by the Vanderbilt Digestive Disease Research Center (DK058404).

1. Reagents and Supplies
Prior to initiating this experimental protocol, prepare the following reagents:
<ol>
 <li>70% ethanol</li>
 <li>1X PBS</li>
 <li>1X DPBS (without Ca and Mg) supplemented with 0.5% BSA</li>
 <li>FACS buffer: 1X DPBS (without Ca and Mg), 2 mM EDTA, and 1% FCS</li>
 <li>ACK buffer: 150 mM NH4Cl, 10 mM KHCO3, and 0.1 mM Na2EDTA in water</li>
</ol>
2. Harvesting and Preparation of Adipose Tissue
<ol>
 <li>Euthanize mice according to IACUC-approved procedures specific to each institution.</li>
 <li>Thoroughly wet the fur with 70% ethanol.</li>
 <li>Make an incision at the level of the xiphoid process (lower part of the sternum) and open the thoracic cavity to expose the heart, taking care not to sever any major blood vessels.</li>
</ol>
Note: At this point, it is also helpful to leave the diaphragm intact as much as possible and to cut a notch out of the right side of the rib cage to allow blood and perfusate to flow out of the thoracic cavity.
<ol start="4">
 <li>Clip the right atrium to allow blood and perfusate to escape the circulatory system.</li>
</ol>
Note: When dealing with obese mice, excess pericardial AT may need to be removed to permit access to the heart.
<ol start="5">
 <li>Grasp the heart with forceps and gently insert a needle into the left ventricle through the apex. Slowly perfuse the mouse with 15 ml sterile PBS.</li>
</ol>
Note: Reduce the rate of perfusion if the lungs begin to fill and expand.
<ol start="6">
 <li>Open the peritoneal cavity and remove the perigonadal fat pads using care to avoid any gonadal tissues.</li>
 <li>Place fat pads in a weigh boat on ice containing 2 ml 1X DPBS (without Mg or Ca) supplemented with 0.5% BSA, and mince the AT into fine pieces.</li>
</ol>
Note: Limit the amount of AT per weigh boat to 1.2 g. If the amount of AT exceeds 1.2 g, divide it evenly between two weigh boats.
<ol start="8">
 <li>Keep AT samples on ice and prepare 3 ml collagenase digest solution per AT sample consisting of 1X DPBS supplemented with 0.5% BSA, 10 mM CaCl2, and 4 mg/ml collagenase, type II.</li>
</ol>
3. Collagenase Digestion
<ol>
 <li>Transfer AT to 50 ml conical tubes by pouring the homogenate and rinsing the weigh boat with 1 ml DPBS (0.5% BSA) and 3 ml collagenase II digest solution.</li>
 <li>Incubate AT homogenate in a rotational shaker (200 rpm) at 37 &deg;C for 20 min.</li>
 <li>Add 10 ml DPBS (0.5% BSA) to conical tubes and place on ice.</li>
 <li>Triturate homogenate numerous times using a 10 ml serological pipette, and pass cell suspensions through 100 &mu;m filter in to a new 50 ml conical tube.</li>
 <li>Centrifuge cell suspension at 500 x g for 10 min at 4 &deg;C.</li>
 <li>Decant supernatant and resuspend SVF cell pellet in 3 ml ACK buffer to lyse contaminating erythrocytes.</li>
 <li>Add 12 ml FACS buffer and centrifuge cell suspension at 500 x g for 10 min at 4 &deg;C.</li>
 <li>Decant supernatant and resuspend SVF cell pellet in FACS buffer.</li>
</ol>
Note: Use the size of the cell pellet as a guide for how much FACS buffer to resuspend in. If the cell pellet covers the bottom of the 50 ml conical tube, use 0.5-1 ml FACS buffer; otherwise, resuspend in 0.25-0.5 ml.
<ol start="9">
 <li>Place samples on ice and prepare 1:10 dilution aliquots of each sample for cell counting by mixing 40 &mu;l FACS buffer, 50 &mu;l trypan blue solution (0.2%), and 10 &mu;l cell suspension.</li>
 <li>Count viable cells based on trypan blue exclusion and dilute cell suspensions to a final concentration of 5-10 x 106 cells/ml.</li>
</ol>
4. Staining of Cell Surface Antigens
<ol>
 <li>Add anti-mouse CD16/CD32 antibody (Fc block) to a final concentration of 0.5-1 &mu;g/106 cells, and incubate on ice for 10 min.</li>
 <li>Transfer samples (&ge;106 cells) to 12 x 75 mm polystyrene round bottom tubes. Prepare separate tubes if analyzing ATMs and T cells, and combine extra cells to prepare an adequate number of tubes to accommodate the required compensation and modified fluorescence minus one (FMO) controls.</li>
</ol>
Note: As an example, when quantifying the proportion of ATMs based on F4/80 and CD11b, the following compensation and FMO controls will need to be prepared:
- Unstained (cells)
- DAPI or propidium iodide (PI) single stain (cells; do not add viability dye until step 5.1)
- F4/80 APC single stain (cells or compensation beads)
- CD11b FITC single stain (cells or compensation beads)
- FMO 1 (cells): Rat IgG2a &kappa; isotype control APC + CD11b FITC + viability dye (added at step 5.1)
- FMO 2 (cells): F4/80 APC + Rat IgG2b &kappa; isotype control FITC + viability dye (added at step 5.1)
<ol start="3">
 <li>Add fluorophore-conjugated primary antibodies and/or isotype controls at the appropriate concentration (see Table of reagents and materials).</li>
 <li>Protect samples from light and incubate at 4 &deg;C for 30 min.</li>
 <li>Add 2 ml FACS buffer and centrifuge cell suspension at 500 x g for 5 min at 4 &deg;C.</li>
 <li>Decant supernatant and resuspend SVF cell pellet in 2 ml FACS buffer.</li>
 <li>Centrifuge cell suspension 500 x g for 5 min at 4 &deg;C, and resuspend SVF cell pellet in &ge;400 &mu;l FACS buffer.</li>
 <li>Transfer samples to 12 x 75 mm polystyrene round bottom tubes equipped with a 35 &mu;m cell strainer tube tops.</li>
 <li>Protect from light, and store samples at 4 &deg;C until FACS analysis.</li>
</ol>
Note: For optimum results cells should be analyzed immeadiately; however, FACS analysis with this protocol has been successfully conducted on labeled cells stored in FACS buffer for 1-2 hr at 4 &deg;C. If cells need to be fixed to increase storage time, labeled cells can be fixed with 2% paraformaldehyde at 4 &deg;C for 24 hr prior to FACS analysis. Antibody companies suggest that labeled cells can be stored for up to one week; however, this has not been tested within the context of this procedure.
5. FACS Analysis
<ol>
 <li>Prior to FACS analysis, add viability dye to samples and appropriate controls to allow for live/dead cell discrimination.</li>
</ol>
Note: Numerous viability dyes are commercially available, but DAPI and PI are recommended depending on the excitation/emission profiles of the fluorophore-conjugated antibodies being used. DAPI and Propidium iodide are added to each sample at a final concentration of 0.2 mg/ml.
<ol start="2">
 <li>Use an unstained negative control sample, preferably cells isolated from the same tissue as the experimental samples, to adjust side scatter (SSC) and forward scatter (FSC) so that the cell population(s) of interest are on scale.</li>
</ol>
Note: For the analysis of AT SVF cells, it is recommended that SSC be displayed in a log scale versus FSC in a linear scale. The use of a log scale for SSC is especially important when analyzing ATMs, which are often very large and granular.
<ol start="3">
 <li>Draw an initial light scatter gate based on the type of cell(s) being analyzed, and adjust the photomultiplier tube (PMT) gain so that the unstained cells are on the far left of a single-parameter histogram (approximately centered on 102) for the appropriate channels.</li>
</ol>
Note: It is recommended that lymphocytes and macrophages (or other myeloid cells) be analyzed separately due to differences in autofluorescence.
<ol start="4">
 <li>Use single stained controls or antibody capture compensation beads to perform multi-color compensation.</li>
</ol>
Note: The use of compensation beads is recommended; however, compatibility of each antibody must be ensured. For example, compensation beads may not cross-react with rabbit antibodies, in which case isolated cells are required to obtain an appropriate single stain control.
<ol start="5">
 <li>Set experimental gates based on modified FMO controls.</li>
 <li>Set the flow cytometer to collect the appropriate number of events based on the prevalence of the population of interest and record experimental data.</li>
 <li>Export FCS data files for offline analysis. There are numerous programs available for flow cytometry data analysis. We recommend Cytobank, a web-based platform that allows investigatores to store and analyze data and generate figures from any computer with internet access 18. Additionally, Cytobank offers the ability to make data public accessible or restrict access to collaborators.</li>
</ol>

<ol>
	<li>Rodbell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+Isolated+Fat+Cells.+I.+Effects+of+Hormones+on+Glucose+Metabolism+and+Lipolysis.">Metabolism of Isolated Fat Cells. I. Effects of Hormones on Glucose Metabolism and Lipolysis.</a> The Journal of Biological Chemistry. 239, 375-380 (1964).</li><li>Danse, L. H., Verschuren, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish+oil-induced+yellow+fat+disease+in+rats.+III.+Lipolysis+in+affected+adipose+tissue.">Fish oil-induced yellow fat disease in rats. III. Lipolysis in affected adipose tissue.</a> Veterinary Pathology. 15, 544-548 (1978).</li><li>Weisberg, S. P., 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=Obesity+is+associated+with+macrophage+accumulation+in+adipose+tissue.">Obesity is associated with macrophage accumulation in adipose tissue.</a> The Journal of Clinical Investigation. 112, 1796-1808 (2003).</li><li>Xu, H., 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=Chronic+inflammation+in+fat+plays+a+crucial+role+in+the+development+of+obesity-related+insulin+resistance.">Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance.</a> The Journal of Clinical Investigation. 112, 1821-1830 (2003).</li><li>Mathis, D., Shoelson, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunometabolism:+an+emerging+frontier.">Immunometabolism: an emerging frontier.</a> Nature Reviews. Immunology. 11, 81 (2011).</li><li>Stefanovic-Racic, 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=Dendritic+cells+promote+macrophage+infiltration+and+comprise+a+substantial+proportion+of+obesity-associated+increases+in+CD11c++cells+in+adipose+tissue+and+liver.">Dendritic cells promote macrophage infiltration and comprise a substantial proportion of obesity-associated increases in CD11c+ cells in adipose tissue and liver.</a> Diabetes. 61, 2330-2339 (2012).</li><li>Liu, 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=Genetic+deficiency+and+pharmacological+stabilization+of+mast+cells+reduce+diet-induced+obesity+and+diabetes+in+mice.">Genetic deficiency and pharmacological stabilization of mast cells reduce diet-induced obesity and diabetes in mice.</a> Nature Medicine. 15, 940-945 (2009).</li><li>Feuerer, 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=Lean,+but+not+obese,+fat+is+enriched+for+a+unique+population+of+regulatory+T+cells+that+affect+metabolic+parameters.">Lean, but not obese, fat is enriched for a unique population of regulatory T cells that affect metabolic parameters.</a> Nature Medicine. 15, 930-939 (2009).</li><li>Nishimura, 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=CD8++effector+T+cells+contribute+to+macrophage+recruitment+and+adipose+tissue+inflammation+in+obesity.">CD8+ effector T cells contribute to macrophage recruitment and adipose tissue inflammation in obesity.</a> Nature Medicine. 15, 914-920 (2009).</li><li>Winer, 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=Normalization+of+obesity-associated+insulin+resistance+through+immunotherapy.">Normalization of obesity-associated insulin resistance through immunotherapy.</a> Nature Medicine. 15, 921-929 (2009).</li><li>Winer, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cells+promote+insulin+resistance+through+modulation+of+T+cells+and+production+of+pathogenic+IgG+antibodies.">B cells promote insulin resistance through modulation of T cells and production of pathogenic IgG antibodies.</a> Nature Medicine. 17, 610-617 (2011).</li><li>Wu, 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=Activation+of+invariant+natural+killer+T+cells+by+lipid+excess+promotes+tissue+inflammation,+insulin+resistance,+and+hepatic+steatosis+in+obese+mice.">Activation of invariant natural killer T cells by lipid excess promotes tissue inflammation, insulin resistance, and hepatic steatosis in obese mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 109. , 1143-1152 (2012).</li><li>Wu, 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=Eosinophils+sustain+adipose+alternatively+activated+macrophages+associated+with+glucose+homeostasis.">Eosinophils sustain adipose alternatively activated macrophages associated with glucose homeostasis.</a> Science. 332, 243-247 (2011).</li><li>Elgazar-Carmon, V., Rudich, A., Hadad, N., Levy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+transiently+infiltrate+intra-abdominal+fat+early+in+the+course+of+high-fat+feeding.">Neutrophils transiently infiltrate intra-abdominal fat early in the course of high-fat feeding.</a> Journal of Lipid Research. 49, 1894-1903 (2008).</li><li>Talukdar, 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=Neutrophils+mediate+insulin+resistance+in+mice+fed+a+high-fat+diet+through+secreted+elastase.">Neutrophils mediate insulin resistance in mice fed a high-fat diet through secreted elastase.</a> Nature Medicine. 18, 1407-1412 (2012).</li><li>Hagman, D. 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=Characterizing+and+quantifying+leukocyte+populations+in+human+adipose+tissue:+impact+of+enzymatic+tissue+processing.">Characterizing and quantifying leukocyte populations in human adipose tissue: impact of enzymatic tissue processing.</a> Journal of Immunological Methods. 386, 50-59 (2012).</li><li>Basu, S., Campbell, H. M., Dittel, B. N., Ray, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+specific+cell+population+by+fluorescence+activated+cell+sorting+(FACS).">Purification of specific cell population by fluorescence activated cell sorting (FACS).</a> J. Vis. Exp. (41), e1546 (2010).</li><li>Kotecha, N., Krutzik, P. O., Irish, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Web-based+analysis+and+publication+of+flow+cytometry+experiments.">Web-based analysis and publication of flow cytometry experiments.</a> Current Protocols in Cytometry. Chapter 10, Unit 10 (2010).</li><li>Cho, C. H., 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=Angiogenic+role+of+LYVE-1-positive+macrophages+in+adipose+tissue.">Angiogenic role of LYVE-1-positive macrophages in adipose tissue.</a> Circulation Research. 100, e47-e57 (2007).</li><li>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=Spectral+compensation+for+flow+cytometry:+visualization+artifacts,+limitations,+and+caveats.">Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats.</a> Cytometry. 45, 194-205 (2001).</li></ol>

We have nothing to disclose.

Increasing interest in the role of the immune system in the metabolic consequences of obesity has led to the widespread use of flow cytometry to characterize immune cells of the AT. Although the exact protocol will vary between laboratories based on their own experience and available equipment, the critical steps include collagenase digestion, differential centrifugation, and cell surface antigen labeling. The goal of the present article is to provide a detailed protocol and practical guide for the isolation of the AT SV...

Historically, the adipose tissue (AT) has been viewed as an inert organ of lipid storage, which expands and contracts in response to energy balance. We now understand that AT represents a dynamic endocrine organ that actively secretes a number of hormones, which directly influence feeding behavior and systemic glucose homeostasis. In addition, over the past decade there has been an increasing appreciation for the numerous populations of immune cells residing in the AT stromal vascular fraction (SVF), as well as their contribution to AT homeostasis.	
 The ability to separate the AT adipocyte and SVF using a collagenase digest followed by differential centrifugation was first described by Rodbell in 1964 1. Collagenase II is most often used for adipocyte and SVF separation due to maintenance of adipocyte insulin receptors1. Early on, enzymatic fractionation of AT was primarily employed to study adipocyte metabolism and to isolate preadipocytes. More recently, this technique, combined with the widespread availability of flow cytometers and the ever-increasing number of commercially available fluorophore-conjugated antibodies, has facilitated the characterization of AT immune cells.	
 Although the presence of immune cells in inflamed AT had been described previously 2, the seminal papers by Weisberg et al. and Xu et al. published in 2003 were the first to document the accumulation of AT macrophages (ATMs) in obesity, which secrete inflammatory cytokines and correlate with AT-specific and systemic insulin resistance 3,4. These observations served as the basis of a new field of investigation recently coined, "immunometabolism," 5 and have been followed up by studies implicating various immune cell populations, including dendritic cells 6, mast cells 7, T cells 8-10, B cells 11, NKT cells 12, eosinophils 13, and neutrophils 14,15 in the development of obesity associated insulin resistance.	The goal of this article is to provide a detailed description of the collagenase digest technique used to isolate cells of the AT SVF and to characterize ATMs and AT T cells via flow cytometry. This protocol has been optimized for mouse AT; however, viewers may benefit from reading an excellent article providing extensive detail on optimization of this technique for human AT 16. The target audience of this article includes investigators with limited experience working with mouse AT and performing flow cytometry. Several practical considerations for balancing cellular yield and viability with time and resources are presented as well as optimal flow cytometry controls for characterizing AT immune cell populations. In addition to our protocol, readers are referred to a recent JoVE article by Basu et al. for an excellent discussion of some of the technical aspects of flow cytometry to include proper controls and compensations 17.

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog #</td>
 <td></td>
 </tr>
 <tr>
 <td>DPBS (no Ca or Mg) 10 x 500 ml</td>
 <td>Life Technologies</td>
 <td>14190-250</td>
 <td></td>
 </tr>
 <tr>
 <td>DAPI</td>
 <td>Life Technologies</td>
 <td>D3571</td>
 <td></td>
 </tr>
 <tr>
 <td>BSA</td>
 <td>Sigma</td>
 <td>A2153-100G</td>
 <td></td>
 </tr>
 <tr>
 <td>collagenase</td>
 <td>Sigma</td>
 <td>C6885-5G</td>
 <td></td>
 </tr>
 <tr>
 <td>propidium iodide solution</td>
 <td>Sigma</td>
 <td>P4864-10 ml</td>
 <td></td>
 </tr>
 <tr>
 <td>stable stack 20 microliter</td>
 <td>Rainin</td>
 <td>SS-L10</td>
 <td></td>
 </tr>
 <tr>
 <td>20 microliter filter tips</td>
 <td>Rainin</td>
 <td>SRL10F</td>
 <td></td>
 </tr>
 <tr>
 <td>stable stack 250 microliter tips</td>
 <td>Rainin</td>
 <td>SS-L250</td>
 <td></td>
 </tr>
 <tr>
 <td>1000 microliter tips</td>
 <td>Rainin</td>
 <td>GPS-L1000</td>
 <td></td>
 </tr>
 <tr>
 <td>1000 microliter filter tips</td>
 <td>Rainin</td>
 <td>GP-L1000F</td>
 <td></td>
 </tr>
 <tr>
 <td>250 microliter Filter Tips</td>
 <td>Rainin</td>
 <td>SR-L200F</td>
 <td></td>
 </tr>
 <tr>
 <td>FC Block</td>
 <td>BD Biosciences</td>
 <td>553142</td>
 <td></td>
 </tr>
 <tr>
 <td>fisher 100mn strainers</td>
 <td>Fisherbrand</td>
 <td>22-363-549</td>
 <td></td>
 </tr>
 <tr>
 <td>medium weigh dish</td>
 <td>Fisherbrand</td>
 <td>02-202B</td>
 <td></td>
 </tr>
 <tr>
 <td>aluminum foil</td>
 <td>Fisherbrand</td>
 <td>1213100</td>
 <td></td>
 </tr>
 <tr>
 <td>mincing scissors</td>
 <td>Fisherbrand</td>
 <td>089531B</td>
 <td></td>
 </tr>
 <tr>
 <td>Vortex</td>
 <td>Fisherbrand</td>
 <td>2215365</td>
 <td></td>
 </tr>
 <tr>
 <td>50 ml conical tubes</td>
 <td>BD Falcon</td>
 <td>14-959-49A</td>
 <td></td>
 </tr>
 <tr>
 <td>filter top FACS tubes</td>
 <td>BD Falcon</td>
 <td>352235</td>
 <td></td>
 </tr>
 <tr>
 <td>10 ml pipette case 200</td>
 <td>BD Falcon</td>
 <td>1367520</td>
 <td></td>
 </tr>
 <tr>
 <td>round bottom tubes</td>
 <td>BD Falcon</td>
 <td>352058</td>
 <td></td>
 </tr>
 <tr>
 <td>5 ml syringe</td>
 <td>BD Falcon</td>
 <td>309646</td>
 <td></td>
 </tr>
 <tr>
 <td>V Bottom Plates</td>
 <td>Costar</td>
 <td>07-200-107</td>
 <td></td>
 </tr>
 <tr>
 <td>transfer bulb pipette</td>
 <td>Thermo Scientific</td>
 <td>13-711-22</td>
 <td></td>
 </tr>
 <tr>
 <td>Shaker</td>
 <td>Thermo Scientific</td>
 <td>11 676 071</td>
 <td></td>
 </tr>
 <tr>
 <td>Adhesive mat</td>
 <td>Thermo Scientific</td>
 <td>1368750</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell Culture Centrifuge</td>
 <td>Sorvall</td>
 <td>75253839</td>
 <td></td>
 </tr>
 <tr>
 <td>Adapters</td>
 <td>Sorvall</td>
 <td>75003723</td>
 <td></td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD16/CD32</td>
 <td>BD Biosciences</td>
 <td>553142</td>
 <td>Concentration: 0.5 - 1 &mu;g/ 106 cells</td>
 </tr>
 <tr>
 <td>Rat anti-mouse F4/80</td>
 <td>eBioscience</td>
 <td>17-4801</td>
 <td>Fluorophore conjugate: APC 
 Concentration: 0.2 &mu;g/ 106 cells 
 Isotype control catalog number: 17-4321</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD11b</td>
 <td>eBioscience</td>
 <td>11-0112</td>
 <td>Fluorophore conjugate: FITC 
 Concentration: 0.5 &mu;g/ 106 cells 
 Isotype control catalog number: 11/1/4031</td>
 </tr>
 <tr>
 <td>Armenian Hamster anti-mouse CD11c</td>
 <td>eBioscience</td>
 <td>12-0114</td>
 <td>Fluorophore conjugate: PE 
 Concentration: 0.8 &mu;g/ 106 cells 
 Isotype control catalog number: Dec-88</td>
 </tr>
 <tr>
 <td>Goat anti-mouse MGL1/2</td>
 <td>R&amp;D Systems</td>
 <td>FAB4297P</td>
 <td>Fluorophore conjugate: PE 
 Concentration: 0.1 &mu;g/ 106 cells 
 Isotype control catalog number: IC108P</td>
 </tr>
 <tr>
 <td>Goat anti-mouse CD206</td>
 <td>R&amp;D Systems</td>
 <td>FAB2535P</td>
 <td>Fluorophore conjugate: PE 
 Concentration: 0.1 &mu;g/ 106 cells 
 Isotype control catalog number: IC108P</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CCR2</td>
 <td>R&amp;D Systems</td>
 <td>FAB5538P</td>
 <td>Fluorophore conjugate: PE 
 Concentration: 0.1 &mu;g/ 106 cells 
 Isotype control catalog number: IC013P</td>
 </tr>
 <tr>
 <td>Armenian Hamster anti-mouse TCR&beta;</td>
 <td>BD Biosciences</td>
 <td>553174</td>
 <td>Fluorophore conjugate: APC 
 Concentration: 0.2 &mu;g/ 106 cells 
 Isotype control catalog number: 553956</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD8a</td>
 <td>BD Biosciences</td>
 <td>552877</td>
 <td>Fluorophore conjugate: PE-Cy7 
 Concentration: 0.8 &mu;g/ 106 cells 
 Isotype control catalog number: 552784</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD4</td>
 <td>BD Biosciences</td>
 <td>557956</td>
 <td>Fluorophore conjugate: Alexa Fluor 700 
 Concentration: 0.2 &mu;g/ 106 cells 
 Isotype control catalog number: 557963</td>
 </tr>
 </tbody>
</table>
Table 1. List of Materials and Reagents.

isolation of adipose tissue immune cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Collagenase digestion of AT followed by differential centrifugation was used to isolate the SVF from epididymal fat pads of male C57BL/6J mice fed a low fat (10% kcal from fat) or high fat (60% kcal from fat) diet (LFD and HFD, respectively) for 16 weeks. Cells of the SVF were then labeled with fluorophore-conjugated primary antibodies to quantify the proportion of viable ATMs (Figure 1) and AT T cells (Figure 2) via FACS analysis. Initial gating, including light scatter, doublet ...

Watch this Scientific Journal Video about Isolation of Adipose Tissue Immune Cells at JoVE.com

Isolation of Adipose Tissue Immune Cells

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Research

JoVE Journal

Immunology and Infection

35.8K Views.  Vanderbilt University School of Medicine. Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.