Name: isolation of adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal adipose depots
Uploaded: 2020-08-16T14:00:00
Description: Read this Scientific Article Protocol about Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots at JoVE.com

The authors are grateful to Lisa Hansen and Kirsten Vestergaard for excellent technical assistance, and P. Scherer, N. Joffin, and C. Crewe for critical reading of the manuscript. The authors thank the UTSW Flow Cytometry Core for excellent guidance and assistance in developing the protocols described here. R.K.G. is supported by NIH NIDDK R01 DK104789, NIDDK RC2 DK118620, and NIDDK R01 DK119163. J.P. is sponsored by a pre-doctoral award from Innovation Fund Denmark.

All animal protocols and procedures have been approved by the University of Texas Southwestern Medical Center Institutional Animal Use and Care Committee.
1. Isolation of stromal vascular fraction (SVF) from gonadal white adipose tissue
<ol>
	<li>Dissect the gonadal white adipose tissue from 6-8-week-old mice and place fat pads in 1x PBS solution.</li>
	<li>Combine up to 4 fat depots (2-4 depots from 1-2 mice recommended) and mince the tissue in a 10 mL beaker containing 200 &#181;L of diges...

<ol>
	<li>Ouchi, N., Parker, J. L., Lugus, J. J., Walsh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipokines+in+inflammation+and+metabolic+disease.">Adipokines in inflammation and metabolic disease.</a> Nature Reviews Immunology. 11 (2), 85-97 (2011).</li><li>Rosen, E. D., Spiegelman, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+we+talk+about+when+we+talk+about+fat.">What we talk about when we talk about fat.</a> Cell. 156 (1-2), 20-44 (2014).</li><li>Funcke, J. B., Scherer, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+adiponectin+and+leptin:+adipose+tissue-derived+mediators+of+inter-organ+communication.">Beyond adiponectin and leptin: adipose tissue-derived mediators of inter-organ communication.</a> Journal of Lipid Research. 60 (10), 1648-1684 (2019).</li><li>Eto, 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=Characterization+of+structure+and+cellular+components+of+aspirated+and+excised+adipose+tissue.">Characterization of structure and cellular components of aspirated and excised adipose tissue.</a> Plastic and Reconstructive Surgery. 124 (4), 1087-1097 (2009).</li><li>Hirsch, J., Batchelor, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+cellularity+in+human+obesity.">Adipose tissue cellularity in human obesity.</a> Clinics in Endocrinology and Metabolism. 5 (2), 299-311 (1976).</li><li>Ghaben, A. L., Scherer, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipogenesis+and+metabolic+health.">Adipogenesis and metabolic health.</a> Nature Reviews Molecular Cell Biology. 20 (4), 242-258 (2019).</li><li>Hepler, C., Gupta, R. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+remodeling+and+obesity.">Adipose tissue remodeling and obesity.</a> Journal of Clinical Investigation. 121 (6), 2094-2101 (2011).</li><li>Kloting, N., 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=Insulin-sensitive+obesity.">Insulin-sensitive obesity.</a> American Journal of Physiology-Endocrinology and Metabolism. 299 (3), 506-515 (2010).</li><li>Vishvanath, L., Gupta, R. 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C., 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+stromal+cell+population+that+inhibits+adipogenesis+in+mammalian+fat+depots.">A stromal cell population that inhibits adipogenesis in mammalian fat depots.</a> Nature. 559 (7712), 103-108 (2018).</li><li>Bagchi, D. P., MacDougald, O. 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+Dissection+of+Diverse+Mouse+Adipose+Depots.">Identification and Dissection of Diverse Mouse Adipose Depots.</a> Journal of Visualized Experiments. (149), e59499 (2019).</li><li>Jeffery, E., Church, C. D., Holtrup, B., Colman, L., Rodeheffer, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+depot-specific+activation+of+adipocyte+precursor+cells+at+the+onset+of+obesity.">Rapid depot-specific activation of adipocyte precursor cells at the onset of obesity.</a> Nature Cell Biology. 17 (4), 376-385 (2015).</li><li>Kim, S. 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=Loss+of+white+adipose+hyperplastic+potential+is+associated+with+enhanced+susceptibility+to+insulin+resistance.">Loss of white adipose hyperplastic potential is associated with enhanced susceptibility to insulin resistance.</a> Cell Metabolism. 20 (6), 1049-1058 (2014).</li><li>Vishvanath, 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=Pdgfrbeta++Mural+Preadipocytes+Contribute+to+Adipocyte+Hyperplasia+Induced+by+High-Fat-Diet+Feeding+and+Prolonged+Cold+Exposure+in+Adult+Mice.">Pdgfrbeta+ Mural Preadipocytes Contribute to Adipocyte Hyperplasia Induced by High-Fat-Diet Feeding and Prolonged Cold Exposure in Adult Mice.</a> Cell Metabolism. 23 (2), 350-359 (2016).</li><li>Wang, Q. A., Tao, C., Gupta, R. K., Scherer, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+adipogenesis+during+white+adipose+tissue+development,+expansion+and+regeneration.">Tracking adipogenesis during white adipose tissue development, expansion and regeneration.</a> Nature Medicine. 19 (10), 1338-1344 (2013).</li><li>Gao, Z., Daquinag, A. C., Su, F., Snyder, B., Kolonin, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDGFRalpha/PDGFRbeta+signaling+balance+modulates+progenitor+cell+differentiation+into+white+and+beige+adipocytes.">PDGFRalpha/PDGFRbeta signaling balance modulates progenitor cell differentiation into white and beige adipocytes.</a> Development. 145 (1), 155861 (2018).</li><li>Rodeheffer, M. S., Birsoy, K., Friedman, 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=Identification+of+white+adipocyte+progenitor+cells+in+vivo.">Identification of white adipocyte progenitor cells in vivo.</a> Cell. 135 (2), 240-249 (2008).</li><li>Church, C. D., Berry, R., Rodeheffer, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+study+of+adipocyte+precursors.">Isolation and study of adipocyte precursors.</a> Methods Enzymol. 537, 31-46 (2014).</li><li>Buffolo, 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=Identification+of+a+Paracrine+Signaling+Mechanism+Linking+CD34(high)+Progenitors+to+the+Regulation+of+Visceral+Fat+Expansion+and+Remodeling.">Identification of a Paracrine Signaling Mechanism Linking CD34(high) Progenitors to the Regulation of Visceral Fat Expansion and Remodeling.</a> Cell Reports. 29 (2), 270-282 (2019).</li><li>Shao, 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=De+novo+adipocyte+differentiation+from+Pdgfrbeta(+)+preadipocytes+protects+against+pathologic+visceral+adipose+expansion+in+obesity.">De novo adipocyte differentiation from Pdgfrbeta(+) preadipocytes protects against pathologic visceral adipose expansion in obesity.</a> Nature Communications. 9 (1), 890 (2018).</li><li>Lee, P. Y., Wang, J. X., Parisini, E., Dascher, C. C., Nigrovic, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ly6+family+proteins+in+neutrophil+biology.">Ly6 family proteins in neutrophil biology.</a> Journal of Leukocyte Biology. 94 (4), 585-594 (2013).</li><li>Vijay, 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=Single-cell+analysis+of+human+adipose+tissue+identifies+depot+and+disease+specific+cell+types.">Single-cell analysis of human adipose tissue identifies depot and disease specific cell types.</a> Nature Metabolism. 2 (1), 97-109 (2020).</li></ol>

The C57BL/6 strain of mice is the most used mouse strain in studies of diet-induced obesity. C57BL/6 mice rapidly gain weight when placed on a high-fat diet (HFD) and develop some of the prominent features of metabolic syndrome associated with obesity (e.g., insulin resistance and hyperlipidemia). Notably, WAT expansion occurring in association with high-fat diet (HFD) feeding occurs in a depot-specific manner19,20,21,</sup...

White adipose tissue (WAT) represents the principal site for energy storage in mammals. Within this tissue, adipocytes, or &#8220;fat cells,&#8221; store excess calories in the form of triglyceride, packaged into large unilocular lipid droplets. Moreover, adipocytes secrete a multitude of factors that regulate various aspects of energy homeostasis1,2,3. Adipocytes constitute the bulk of WAT volume; however, adipocytes only represent less than 50% of total cells found in WAT4,5. The non-adipocyte compartment of WAT, or stromal-vascular fraction (SVF), is quite heterogeneous and contains vascular endothelial cells, tissue-resident immune cells, fibroblasts, and adipocyte precursor cell (APC) populations.
WAT is exceptional in its remarkable capacity to expand in size as the demand for energy storage increases. Maintaining this tissue plasticity is essential as adequate storage of lipids in WAT protects against deleterious ectopic lipid deposition into non-adipose tissues6. The manner by which individual WAT depots undergo this expansion in response to caloric excess is a critical determinant of insulin sensitivity in the setting of obesity7. Pathologic WAT expansion, observed in obese individuals with metabolic syndrome, is characterized by preferential expansion of visceral WAT depots at the expense of metabolically favorable subcutaneous fat tissue. Moreover, insulin resistance in obesity is associated with pathologic remodeling of WAT. This is characterized by hypertrophic growth of existing adipocytes (increase in size), inadequate angiogenesis, chronic metabolic-inflammation, accumulation of extracellular matrix components (fibrosis), and tissue hypoxia8,9. These WAT phenotypes of obesity are associated with hepatic steatosis and insulin resistance, similar to what is observed in the condition of lipodystrophy (absence of functional WAT). In contrast, healthy WAT expansion is observed in the metabolically healthy obese population and is characterized by preferential expansion of protective subcutaneous WAT and depot expansion through adipocyte hyperplasia10. The recruitment of new adipocytes is mediated by de novo adipocyte differentiation from adipocyte precursor cells (APCs) (termed, &#8220;adipogenesis&#8221;). Adipocyte hyperplasia coincides with relatively lower degrees of WAT fibrosis and metabolic inflammation6,11. A multitude of cell types within the WAT microenvironment directly influence the health and expandability of WAT in obesity12. As such, defining the function of the various cell types present in WAT remains a high priority for the field.
Over the past decade, several strategies have been employed to define and isolate native APCs from human and mouse WAT SVF13. Such strategies isolate APCs based on the cell surface expression of common mesenchymal stem/progenitor cell markers using antibody-based cell separation techniques. These approaches include fluorescence-activated cell sorting (FACS), using fluorophore-labelled antibodies, or immunomagnetic bead separation (i.e., chemically modified antibodies). Cell surface proteins targeted for the isolation of APCs include PDGFR&#945;, PDGFR&#946;, CD34, and SCA-1. These approaches have helped enrich for APCs; however, cell populations isolated based on these markers are quite heterogeneous. Very recent single-cell RNA-sequencing (scRNA-seq) studies have highlighted the molecular and functional heterogeneity of stromal cells within the isolated stromal-vascular fraction (SVF) of murine WAT14,15,16,17. From our own scRNA-seq and functional analyses, we have identified and characterized functionally distinct immune-modulating and adipogenic PDGFR&#946;+ perivascular cell subpopulations in the stromal compartment of intra-abdominal WAT in adult mice15. Fibro-inflammatory precursors, or FIPs, represent a prominent subpopulation of PDGFR&#946;+ cells and can be isolated based on LY6C expression (LY6C+ PDGFR&#946;+ cells)15. FIPs lack adipogenic capacity, exert a strong pro-inflammatory response to various stimuli, produce collagen, and secrete anti-adipogenic factors15. The pro-inflammatory and fibrogenic activity of these cells increases in association with obesity in mice, implicating these cells as regulators of WAT remodeling. The LY6C- CD9- PDGFR&#946;+ subpopulation represents adipocyte precursor cells (APCs). These APCs are enriched in the expression of Pparg and other pro-adipogenic genes, and readily differentiate into mature adipocytes in vitro and in vivo15. Here, we provide a detailed protocol for the isolation of these distinct cell populations from intra-abdominal WAT depots of adult mice using FACS, and immunomagnetic bead separation with biotinylated antibodies. This protocol can be used to isolate functionally distinct adipose progenitor subpopulations from multiple intra-abdominal WAT depots of adult male and female mice15. Studying these functionally distinct cell populations in isolation may contribute greatly to our current understanding of the molecular mechanisms that regulate adipogenesis and intra-abdominal adipose tissue remodeling in health and disease.
The protocol below details the isolation of adipose progenitors from murine epididymal WAT; however, the same procedure can be used to isolate corresponding cells from the mesenteric and retroperitoneal WAT depots of both male and female mice15. A detailed protocol on how to identify and isolate these depots in mice can be found in Bagchi et al.18. This protocol has been optimized for the use of mice 6-8 weeks of age. The frequency and differentiation capacity of APCs may decline in association with ageing.

<table>
	<tbody>
		<tr>
			<td>Mechanical Tissue Preparation and SVF Isolation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>40 and 100 &#181;m cell strainers</td>
			<td>Fisher Scientific</td>
			<td>352340/352360</td>
			<td></td>
		</tr>
		<tr>
			<td>1X Phosphate buffered saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>21040CV</td>
			<td></td>
		</tr>
		<tr>
			<td>5ml polypropylene tubes</td>
			<td>Fisher Scientific</td>
			<td>352053</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digestion Buffer (for 10mL)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml HBSS</td>
			<td>Sigma</td>
			<td>H8264</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mg Collagenase D (1 mg/ml final cc.)</td>
			<td>Roche</td>
			<td>11088882001</td>
			<td></td>
		</tr>
		<tr>
			<td>0.15 g BSA (1.5 % final cc.)</td>
			<td>Fisher Scientific</td>
			<td>BP1605-100</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Immunomagnetic separation of APCs and non-APCs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5X MojoSort Buffer (MS buffer)</td>
			<td>BioLegend</td>
			<td>480017</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml MojoSort Magnet (MS magnet)</td>
			<td>BioLegend</td>
			<td>480019</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;L MojoSort Streptavidin Nanobeads</td>
			<td>BioLegend</td>
			<td>480015</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Purity Check and FACS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10X Red Blood Cell Lysis Buffer</td>
			<td>eBioscience</td>
			<td>00-4300-54</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc block (Mouse CD16/CD32)</td>
			<td>eBioscience</td>
			<td>553141</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin CD45</td>
			<td>BioLegend</td>
			<td>103103</td>
			<td>Concentration: &#8804; 0.25 &#181;g per 10^6 cells 
			Species: Mouse 
			Clone: 30-F11</td>
		</tr>
		<tr>
			<td>Biotin CD31</td>
			<td>BioLegend</td>
			<td>102503</td>
			<td>Concentration: &#8804; 0.25 &#181;g per 10^6 cells 
			Species: Mouse 
			Clone: MEC13.3</td>
		</tr>
		<tr>
			<td>Biotin CD9</td>
			<td>BioLegend</td>
			<td>124803</td>
			<td>Concentration: &#8804; 0.25 &#181;g per 10^6 cells 
			Species: Mouse 
			Clone: MZ3</td>
		</tr>
		<tr>
			<td>Biotin LY6C</td>
			<td>BioLegend</td>
			<td>128003</td>
			<td>Concentration: &#8804; 0.25 &#181;g per 10^6 cells 
			Species: Mouse 
			Clone: HK1.4</td>
		</tr>
		<tr>
			<td>CD31-PerCP/Cy5.5</td>
			<td>BioLegend</td>
			<td>102419</td>
			<td>Concentration: Dilution 1:400 
			Species: Mouse 
			Clone: 390</td>
		</tr>
		<tr>
			<td>CD45-PerCP/Cy5.5</td>
			<td>BioLegend</td>
			<td>103131</td>
			<td>Concentration: Dilution 1:400 
			Species: Mouse 
			Clone: 30-F11</td>
		</tr>
		<tr>
			<td>CD140b PDGFR&#946;-PE</td>
			<td>BioLegend</td>
			<td>136006</td>
			<td>Concentration: Dilution 1:50 
			Species: Mouse 
			Clone: APB5</td>
		</tr>
		<tr>
			<td>LY6C-APC</td>
			<td>BioLegend</td>
			<td>128016</td>
			<td>Concentration: Dilution 1:400 
			Species: Mouse 
			Clone: HK1.4</td>
		</tr>
		<tr>
			<td>CD9-FITC</td>
			<td>BioLegend</td>
			<td>124808</td>
			<td>Concentration: Dilution 1:400 
			Species: Mouse 
			Clone: MZ3</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture and Differentiation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gonadal APC Culture media (for 500mL)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>288 mL DMEM with 1 g/L glucose</td>
			<td>Corning</td>
			<td>10-014-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>192 mL MCDB201</td>
			<td>Sigma</td>
			<td>M6770</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Fetal bovine serum (FBS)** lot#14E024</td>
			<td>Sigma</td>
			<td>12303C</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL 100% ITS premix</td>
			<td>BD Bioscience</td>
			<td>354352</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL 10 mM L-ascorbic acid-2-2phosphate</td>
			<td>Sigma</td>
			<td>A8960-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>50 &#181;L 100 g/ml FGF-basic</td>
			<td>R&#38;D systems</td>
			<td>3139-FB-025/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Pen/Strep</td>
			<td>Corning</td>
			<td>30-001-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>500 &#181;L Gentamycin</td>
			<td>Gibco</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr>
			<td>**NOTE: The adipogenic capacity of primary APCs can vary from lot to lot of commercial FBS. Multiple lots/sources of FBS should be tested.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal adipose depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes two strategies that allow for the isolation of distinct stromal cell populations from intra-abdominal WAT depots of adult mice. APCs and FIPs can be isolated by FACS (Figure 1) or immunomagnetic bead separation with biotinylated antibodies (Figure 2). Both approaches utilize reagents and antibodies that are all commercially available. Immunomagnetic bead separation leads to the separation of adipogenic from non-adipogenic cells from the g...

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Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

The protocol allows investigators to isolate functionally and molecularly distinct subpopulations of perigonadal white adipose tissue stromal cells from mouse models of interest. The advantage of this technique is that all tools and reagents are widely available. Demonstrating the procedure will be Lavanya Vishvanath, a senior laboratory research analyst from Dr.Rana Gupta's laboratory.After stromal vascular cell isolation from gonadal white adipose tissue, or WAT, resuspend the cell pellet in one milliliter of red blood cell lysis buffer for one to two minute incubation at room temperature. Stop the lysis with 10 milliliters of 2%FBS and PBS and filter the cells through a 40 micron cell strainer into a new 50 milliliter centrifuge tube. Collect the cells by centrifugation and resuspend the pellet in 400 to 800 microliters FBS and PBS, supplemented with FC block.After a 10 minute incubation at four degrees Celsius, transfer 400 to 800 microliters of cells to the appropriate number of tubes for fluorescence-conjugated antibody staining and label the cells with the control and experimental antibodies of interest at four degrees Celsius for 15 minutes, protected from light. At the end of the incubation, pellet the cells by centrifugation and wash the cells in 400 microliters of fresh FBS and PBS per tube. After the wash, resuspend the pellets in 400 to 800 microliters of fresh FBS and PBS per tube, and filter the cells through 40 micron filter caps into five milliliter polystyrene round bottom tubes for FACS.Set the compensation and experimental gates to obtain the antigen-presenting cells and fibroinflammatory progenitors. After cell sorting, collect and wash the cells by centrifugation, and resuspend the pellets in 500 microliters of gonadal antigen-presenting cell culture medium per fraction. For immunomagnetic bead separation of the adipogenic and non-adipogenic cell fractions, resuspend the stromal vascular pellet in 10 milliliters of MS buffer and filter the cells through a 40 micron strainer.After counting, collect the cells by centrifugation and resuspend the cells at no more than one times 10 to the eighth cells per milliliter of MS buffer concentration. Next, add no more than 0.25 grams of anti-CD31 and anti-CD45 biotin-conjugated antibody per one times ten to the six cells for a 15 minute incubation on ice. At the end of the incubation, wash the cells in four milliliters of MS buffer by centrifugation, and resuspend the pellet in 100 microliters of fresh MS buffer.Add 10 microliters of streptavidin nanobeads to the cells for a 15 minute incubation on ice, followed by another wash in four milliliters of MS buffer. Resuspend the pellet in 2.5 milliliters of fresh MS buffer and transfer the cells to a five milliliter polypropylene tube. Place the tube onto a magnet for five minutes before decanting the unlabeled cells into a new 15 milliliter centrifuge tube.Repeat the wash two more times as demonstrated, pulling the unlabeled cells after each wash, and collect the endothelial and hematopoietic lineage-depleted cells by centrifugation. Resuspend the pellet in 100 microliters MS buffer and label the cells with no more than 0.25 micrograms of Ly6C and CD-9 antibodies per one times 10 of the sixth cells for 15 minutes on ice. At the end of the incubation, collect the unbound Ly6C negative CD-9 negative adipogenic cells as just demonstrated before collecting the Ly6C positive non-adipogenic cells.Then, collect both cell populations by centrifugation and resuspend the cells in 500 microliters of gonadal antigen-presenting cell culture medium per fraction. To set up cell cultures for differentiation, plate each fraction at four times 10 of the fourth cells per well in concentration in individual wells of a 48 well culture plate and place the plates in the cell culture incubator for seven to 10 days. The adipocyte precursor cells will begin to undergo differentiation into adipocytes as they approach confluence, while the fibroin inflammatory progenitors will be resistant to undergoing adipogenesis.Using this protocol, distinct stromal cell populations can be isolated from intraabdominal WAT depots of adult mice by FACS or immunomagnetic bead separation as demonstrated. With immunomagnetic bead separation, adipogenic and non-adipogenic cells can be isolated from the gonadal WAT stromal vascular fraction. As observed by flow cytometry, 75%of the cells within the adipogenic fraction are Ly6C negative CD-9 negative adipocyte precursor cells, while over 75%of the non-adipogenic fraction, Ly6C positive fibroinflammatory progenitor cells.Adipocyte precursor cells isolated either method differentiate into lipid-containing adipocytes to a high purity within seven to 10 days of plating. In contrast, non adipogenic precursors remain fibroblast-like and do not become adipocytes under the same culture conditions. The protocol works for the isolation of adipogenic and fibroinflammatory stromal cell subpopulations from perigonadal white adipose tissue.Similar cell populations can be found in other adipose depots of mice. However, their selection will rely on the use of different antibodies. The cell populations isolated through FACS and magnetic separation can be used for in vitro assays of cell differentiation or further characterized through gene expression analysis.

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