The authors would like to thank Takahito Wada and Saiko Yoshida (The University of Tokyo, Tokyo, Japan) for their experimental assistance. This work was funded by the following grants to Y.H.: research grant from the University of Tokyo Excellent Young Researcher Program; Japan Society for the Promotion of Science (JSPS) KAKENHI Grant-in-Aid for Early-Career Scientists, grant number 19K17976; grant for the Front Runner of Future Diabetes Research (FFDR) from the Japan Foundation for Applied Enzymology, grant number 17F005; grant from the Pharmacological Research Foundation; grant from the Mochida Memorial Foundation for Medical and Pharmaceutical Research; grant from the MSD Life Science Foundation; grant from the Daiwa Securities Health Foundation; grant from the Tokyo Biochemical Research Foundation; Life Science Research grant from the Takeda Science Foundation; and grant from the SENSHIN Medical Research Foundation.

All animal experiments described in this protocol were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Tokyo and performed according to the institutional guidelines of the University of Tokyo.
1. Preparation of enzyme solution and medium
<ol>
	<li>Put both sides of inguinal white adipose tissue (right and left side, approximately 150 mg) from a 7-8-week-old mouse and 2.5 mL of enzyme solution into tube C of the dissociator.</li>
	<li>Reconstitute Enzyme D with 3 mL of Dulbecco&#39;s modified eagle medium (DMEM)/F12 without fetal bovine serum (FBS) or antibiotics, Enzyme R with 2.7 mL of DMEM/F12 without FBS or antibiotics, and Enzyme A with 1 mL of buffer A.</li>
	<li>Prepare 2.5 mL of enzyme solution by adding 100 &#181;L of Enzyme D, 50 &#181;L of Enzyme R, 12.5 &#181;L of Enzyme A, and 2.35 mL of DMEM/F12 without FBS or antibiotics into tube C. Set tube C with enzyme solution on ice.</li>
</ol>
2. Isolation of adipose tissue
<ol>
	<li>Euthanize 7-8-week-old male C57BL/6J mice (approximately 20 g) by cervical dislocation.</li>
	<li>At a clean bench, to isolate inguinal white adipose tissue, cut the skin with blunt/sharp scissors at the abdomen and toward the lower extremities. Isolate the adipose tissue from the inside of the thighs using sharp/sharp scissors. One side of inguinal adipose tissue wieghs ~75 mg.</li>
	<li>Put the isolated adipose tissue into tube C with enzyme solution on ice, and keep the tube within the clean bench to maintain sterility.</li>
</ol>
3. Mincing and digestion of adipose tissue
<ol>
	<li>At the clean bench, add 2.5 mL of enzyme solution to tube C.</li>
	<li>Mince the adipose tissue into small pieces by cutting with sharp/sharp scissors 50 times. Cut the adipose tissue into ~2 mm2 pieces.</li>
	<li>Tightly close the cap of tube C, turn the tube upside down, and attach it to the sleeve of the tissue dissociatorwith the cap on. Then, digest the sample for ~40 min at 37 &#176;C. 
	&#8203;NOTE: This study used gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec 130-096-427), and the preinstalled program &#34;37C_mr_ATDK_1&#34; was followed.</li>
</ol>
4. Filtration of cell suspension
<ol>
	<li>Pre-warm DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin to 37 &#176;C.</li>
	<li>Detach tube C from the tissue dissociator and stop digestion by adding 5 mL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin and gently pipetting four times.</li>
	<li>Centrifuge at 700 x g for 10 min at 20 &#176;C.</li>
	<li>Carefully aspirate the supernatant without disturbing the cell pellet.</li>
	<li>Resuspend the pellet by adding 10 mL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin and gently pipetting five times.</li>
	<li>Filter the cell suspension through a 70 &#181;m diameter cell strainer placed on a new 50 mL tube.</li>
	<li>Centrifuge at 250 x g for 5 min and resuspend the pellet in 10 mL of phosphate-buffered saline (PBS).</li>
</ol>
5. Cell plating
<ol>
	<li>Centrifuge at 500 x g for 5 min.</li>
	<li>Remove the supernatant and resuspend the pellet by adding 10 mL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin and pipetting ten times.</li>
	<li>Plate the cell suspension on a 10 cm collagen-coated dish and place the dishes in a cell culture incubator (37 &#176;C, 5% CO2) for 1-2 h. 
	NOTE: Collagen-coated dishes are not be absolutely required. However, based on experience, collagen-coated dishes do help the adherence of preadipocytes and thus increase the survival of the cells.</li>
	<li>Aspirate the medium and wash the cells twice with 3 mL of PBS per wash.</li>
	<li>Add 10 mL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin and return the dishes to the cell-culture incubator (37 &#176;C, 5% CO2).</li>
</ol>
6. Passaging of preadipocytes
<ol>
	<li>The next day, aspirate the medium (the cells will be adherent, and thus the medium can be aspirated), wash the cells twice with 3 mL of PBS per wash, and add 10 mL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin.</li>
	<li>When the cells reach 80% confluence (usually 4 days after SVF isolation), split the cells into four 10 cm collagen-coated dishes.
	<ol>
		<li>Specifically, aspirate the medium, wash the cells with PBS (room temperature [RT]), add 1 mL of pre-warmed 0.05% trypsin, and incubate for 5 min in the cell-culture incubator.</li>
		<li>Add 10 mL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin to quench the trypsin activity. After pipetting, centrifuge at 440 x g for 5 min.</li>
		<li>Aspirate the supernatant, resuspend the cells by adding 40 mL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin, and plate the cells in four 10 cm collagen-coated dishes.</li>
	</ol>
	</li>
	<li>Passage the cells again when they reach 80% confluence.</li>
</ol>
7. Preparation of retrovirus expressing SV40 large T antigen for immortalization of preadipocytes (optional)
<ol>
	<li>At least 3 days before immortalization, plate Platinum-E (Plat-E) packaging cells29 at a density of 3.0 x 105 cells/mL in 2 mL of DMEM with 10% FBS without antibiotics. Use a hemacytometer to count the cells.</li>
	<li>The next day, transfect 4 &#181;g of pBABE-neo largeTcDNA (add gene plasmid #1780) using Lipofectamine 2000, according to the manufacturer&#39;s instructions.</li>
	<li>After 24 h, aspirate the medium and add 2 mL of DMEM with 10% FBS and 1% penicillin-streptomycin.</li>
	<li>The next day, harvest the retroviral supernatant. The retrovirus can be used for immortalization immediately or stored at -80 &#176;C.</li>
</ol>
8. Immortalization of preadipocytes with SV40 large T antigen (optional)
<ol>
	<li>Passage and expand the preadipocytes twice after SVF isolation.</li>
	<li>Plate the cells in 6-well plates at a density of 0.5-1.0 x 104 cells/mL in 2 mL of DMEM/F12 with 10% FBS and 1% penicillin-streptomycin. Use a hemacytometer to count the cells.</li>
	<li>On the next day, prepare 250 &#181;L of fresh or thawed retrovirus expressing SV40 large T antigen per well of the 6-well plates. Centrifuge at 440 x g for 5 min and place the supernatant in a new tube.</li>
	<li>Add 750 &#181;L of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin and 1 &#181;L of polybrene per 250 &#181;L of retroviral supernatant to make the working virus.</li>
	<li>Aspirate the medium and add 1,000 &#181;L of the working retrovirus to each well containing preadipocytes.</li>
	<li>Add 1 mL of DMEM/F12 with 10% FBS and 1% penicillin-streptomycin 4 h later.</li>
	<li>The next day, separate the infected preadipocytes into a 10 cm dish and select the infected cells with DMEM/F12 containing 10% FBS, 1% penicillin-streptomycin, and 500 &#181;g/mL neomycin. To monitor the antibiotics selection, prepare a dish of uninfected cells with neomycin.</li>
	<li>Continue to passage the infected cells with neomycin until all uninfected cells in the monitor dish are dead.</li>
</ol>
9. Adipocyte differentiation
<ol>
	<li>Plate the cells. For 12-well plates, plate the cells at a density of 4.0 x 104 cells/mL in 1 mL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin.</li>
	<li>When the preadipocytes become confluent (48-72 h after plating), aspirate the medium and replace it with differentiation medium (see Table 1 for composition).</li>
	<li>At day 2 of differentiation (i.e., 48 h after inducing differentiation), replace the medium with maintenance medium (see Table 1 for composition). Afterward, change the maintenance medium every 2 days. Terminal differentiation is achieved at day 7 of differentiation.</li>
	<li>Oil red o staining
	<ol>
		<li>For oil red o staining, aspirate the medium and rinse the cells with 1 mL of PBS per well. Fix the cells by adding 1 mL of 4% formaldehyde per well, and incubate the cells for 15 min at RT.</li>
		<li>During the incubation, dilute the 0.5% oil red o stock solution (in isopropyl alcohol) to 0.3% using ddH2O and filter the working solution using a filter paper.</li>
		<li>After the 15 min of incubation, remove the formaldehyde, rinse the cells with 60% isopropyl alcohol, add 1 mL of filtered oil red o working solution, and incubate for 1 h at RT.</li>
		<li>After the incubation, wash the cells with running water. Then, observe lipid-laden adipocytes under an inverted microscope (magnification: 20x objective and 10x eyepiece).</li>
	</ol>
	</li>
	<li>mRNA expression analysis 
	NOTE: Refer to Table 2 for the list of primers used in this study.
	<ol>
		<li>Aspirate the medium and add 1 mL/well of trizol to the well.</li>
		<li>Incubate for 15 min at RT, detach the cells by pipetting, and collect the samples in 1.5 mL tubes. The samples can be stored at -80 &#176;C until RNA extraction.</li>
		<li>Thaw the frozen sample to RT, add 200 &#181;L of chloroform to the sample, and shake vigorously for 15 s.</li>
		<li>Incubate the sample at RT for 3 min and then spin at 20,000 x g for 15 min at 4 &#176;C.</li>
		<li>Carefully remove the aqueous phase (top, colorless) and transfer to new tubes. Never transfer the protein phase (middle, white) or the phenol/chloroform phase (bottom, pink).</li>
		<li>Slowly add an equal volume of 70% EtOH and mix gently. Do not vortex.</li>
		<li>Load the sample (up to 700 &#181;L) into an RNA spin column seated in a collection tube. Be sure to include any precipitate that may have formed.</li>
		<li>Spin at 9,000 x g for 30 s at 4 &#176;C , and then discard the flow-through.</li>
		<li>Add 700 &#181;L of buffer RW1, spin at 9,000 x g for 30 s at 4 &#176;C , and then discard the flow-through.</li>
		<li>Transfer the column into a new collection tube and add 500 &#181;L of buffer RPE.</li>
		<li>Spin at 9,000 x g for 30 s at 4 &#176;C and then discard the flow-through.</li>
		<li>Add 500 &#181;L of buffer RPE, spin at 9,000 x g for 2 min at 4 &#176;C, and then discard the flow-through.</li>
		<li>Transfer the column to a new collection tube, and spin (columns without buffer) at 9,000 x g for 1-2 min at 4 &#176;C.</li>
		<li>Transfer the column into a new 1.5 mL collection tube and add 30 &#181;L of RNase-free water directly onto the column membrane.</li>
		<li>Incubate the sample at RT for 1-2 min. Then, spin at 9,000 x g for 1 min at 4 &#176;C to elute the RNA.</li>
		<li>Check the RNA concentration using a fluorospectrometer. 
		NOTE: It is recommended to align the concentration here.</li>
		<li>Perform reverse transcription using ~200 ng of RNA template. Use a commercial quantitative real-time polymerase chain reaction (qPCR-RT) kit and follow the manufacturer&#39;s protocol. After the reaction, dilute the cDNA 20 times to 200 &#181;L.</li>
		<li>Add 2.0 &#181;L of cDNA, 3.0 &#181;L of Power Sybr Green Master Mix, 0.018 &#181;L of 100 &#181;M qPCR forward primer, 0.018 &#181;L of 100 &#181;M qPCR reverse primer, and 0.96 &#181;L of ddH2O to each well of a 384-well plate.</li>
		<li>Run a qPCR (hold stage: 50&#176;C for 2 min and 95 &#176;C for 2 min; PCR stage: 40 cycles of 95 &#176;C for 15 s and 60 &#176;C for 1 min; melt curve stage: 95 &#176;C for 15 s, 60 &#176;C for 1 min, and 95 &#176;C for 15 s). Use the standard curve method for quantification.</li>
	</ol>
	</li>
</ol>

Semi-Automated Isolation of Murine White Adipose Tissue

<ol>
	<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>Hotamisligil, G. S., Shargill, N. S., 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=Adipose+expression+of+tumor+necrosis+factor-alpha:+direct+role+in+obesity-linked+insulin+resistance.">Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance.</a> Science. 259 (5091), 87-91 (1993).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positional+cloning+of+the+mouse+obese+gene+and+its+human+homologue.">Positional cloning of the mouse obese gene and its human homologue.</a> Nature. 372 (6505), 425-432 (1994).</li><li>Kajimura, S., Saito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+era+in+brown+adipose+tissue+biology:+Molecular+control+of+brown+fat+development+and+energy+homeostasis.">A new era in brown adipose tissue biology: Molecular control of brown fat development and energy homeostasis.</a> Annual Review of Physiology. 76, 225-249 (2014).</li><li>Wu, 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=Beige+adipocytes+are+a+distinct+type+of+thermogenic+fat+cell+in+mouse+and+human.">Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human.</a> Cell. 150 (2), 366-376 (2012).</li><li>Loos, R. J. F., Yeo, G. S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+obesity:+from+discovery+to+biology.">The genetics of obesity: from discovery to biology.</a> Nature Reviews Genetics. 23 (2), 120-133 (2022).</li><li>Loos, R. J. F., Yeo, G. S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bigger+picture+of+FTO-the+first+GWAS-identified+obesity+gene.">The bigger picture of FTO-the first GWAS-identified obesity gene.</a> Nature Reviews Endocrinology. 10 (1), 51-61 (2014).</li><li>Hiraike, Y., Yang, C. T., Liu, W. J., Yamada, T., Lee, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FTO+obesity+variant-exercise+interaction+on+changes+in+body+weight+and+BMI:+The+Taiwan+biobank+study.">FTO obesity variant-exercise interaction on changes in body weight and BMI: The Taiwan biobank study.</a> The Journal of Clinical Endocrinology and Metabolism. 106 (9), e3673-e3681 (2021).</li><li>Li, 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=Physical+activity+attenuates+the+genetic+predisposition+to+obesity+in+20,000+men+and+women+from+EPIC-Norfolk+prospective+population+study.">Physical activity attenuates the genetic predisposition to obesity in 20,000 men and women from EPIC-Norfolk prospective population study.</a> PLoS Medicine. 7 (8), e1000332 (2010).</li><li>Claussnitzer, 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=FTO+obesity+variant+circuitry+and+adipocyte+browning+in+humans.">FTO obesity variant circuitry and adipocyte browning in humans.</a> The New England Journal of Medicine. 373 (10), 895-907 (2015).</li><li>Tontonoz, P., Hu, E., 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=Stimulation+of+adipogenesis+in+fibroblasts+by+PPAR+gamma+2,+a+lipid-activated+transcription+factor.">Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor.</a> Cell. 79 (7), 1147-1156 (1994).</li><li>Seale, 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=Transcriptional+control+of+brown+fat+determination+by+PRDM16.">Transcriptional control of brown fat determination by PRDM16.</a> Cell Metabolism. 6 (1), 38-54 (2007).</li><li>Seale, 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=PRDM16+controls+a+brown+fat/skeletal+muscle+switch.">PRDM16 controls a brown fat/skeletal muscle switch.</a> Nature. 454 (7207), 961-967 (2008).</li><li>Rajakumari, 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=EBF2+determines+and+maintains+brown+adipocyte+identity.">EBF2 determines and maintains brown adipocyte identity.</a> Cell Metabolism. 17 (4), 562-574 (2013).</li><li>Shapira, S. 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=EBF2+transcriptionally+regulates+brown+adipogenesis+via+the+histone+reader+DPF3+and+the+BAF+chromatin+remodeling+complex.">EBF2 transcriptionally regulates brown adipogenesis via the histone reader DPF3 and the BAF chromatin remodeling complex.</a> Genes and Development. 31 (7), 660-673 (2017).</li><li>Hiraike, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NFIA+co-localizes+with+PPAR&#947;+and+transcriptionally+controls+the+brown+fat+gene+program.">NFIA co-localizes with PPAR&#947; and transcriptionally controls the brown fat gene program.</a> Nature Cell Biology. 19 (9), 1081-1092 (2017).</li><li>Hiraike, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NFIA+differentially+controls+adipogenic+and+myogenic+gene+program+through+distinct+pathways+to+ensure+brown+and+beige+adipocyte+differentiation.">NFIA differentially controls adipogenic and myogenic gene program through distinct pathways to ensure brown and beige adipocyte differentiation.</a> PLoS Genetics. 16 (9), e1009044 (2020).</li><li>Hiraike, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NFIA+determines+the+cis-effect+of+genetic+variation+on+Ucp1+expression+in+murine+thermogenic+adipocytes.">NFIA determines the cis-effect of genetic variation on Ucp1 expression in murine thermogenic adipocytes.</a> iScience. 25 (8), 104729 (2022).</li><li>Villanueva, C. 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=Adipose+subtype-selective+recruitment+of+TLE3+or+Prdm16+by+PPAR&#947;+specifies+lipid+storage+versus+thermogenic+gene+programs.">Adipose subtype-selective recruitment of TLE3 or Prdm16 by PPAR&#947; specifies lipid storage versus thermogenic gene programs.</a> Cell Metabolism. 17 (3), 423-435 (2013).</li><li>Pearson, 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=Loss+of+TLE3+promotes+the+mitochondrial+program+in+beige+adipocytes+and+improves+glucose+metabolism.">Loss of TLE3 promotes the mitochondrial program in beige adipocytes and improves glucose metabolism.</a> Genes and Development. 33 (13-14), 747-762 (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=Zfp423+maintains+white+adipocyte+identity+through+suppression+of+the+beige+cell+thermogenic+gene+program.">Zfp423 maintains white adipocyte identity through suppression of the beige cell thermogenic gene program.</a> Cell Metabolism. 23 (6), 1167-1184 (2016).</li><li>Green, H., Kehinde, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sublines+of+mouse+3T3+cells+that+accumulate+lipid.">Sublines of mouse 3T3 cells that accumulate lipid.</a> Cell. 1 (3), 113-116 (1974).</li><li>Green, H., Kehinde, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+established+preadipose+cell+line+and+its+differentiation+in+culture+II.+Factors+affecting+the+adipose+conversion.">An established preadipose cell line and its differentiation in culture II. Factors affecting the adipose conversion.</a> Cell. 5 (1), 19-27 (1975).</li><li>Green, H., Kehinde, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+heritable+changes+leading+to+increased+adipose+conversion+in+3T3+cells.">Spontaneous heritable changes leading to increased adipose conversion in 3T3 cells.</a> Cell. 7 (1), 105-113 (1976).</li><li>Aune, U. L., Ruiz, L., Kajimura, 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+differentiation+of+stromal+vascular+cells+to+beige/brite+cells.">Isolation and differentiation of stromal vascular cells to beige/brite cells.</a> Journal of Visualized Experiments. (73), e50191 (2013).</li><li>Galmozzi, A., Kok, B. P., Saez, E. <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+differentiation+of+primary+white+and+brown+preadipocytes+from+newborn+mice.">Isolation and differentiation of primary white and brown preadipocytes from newborn mice.</a> Journal of Visualized Experiments. (167), e62005 (2021).</li><li>Priya, N., Sarcar, S., Majumdar, A. S., SundarRaj, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explant+culture:+a+simple,+reproducible,+efficient+and+economic+technique+for+isolation+of+mesenchymal+stromal+cells+from+human+adipose+tissue+and+lipoaspirate.">Explant culture: a simple, reproducible, efficient and economic technique for isolation of mesenchymal stromal cells from human adipose tissue and lipoaspirate.</a> Journal of Tissue Engineering and Regenerative Medicine. 8 (9), 706-716 (2014).</li><li>Lockhart, R. A., Aronowitz, J. A., Dos-Anjos Vilaboa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+freshly+isolated+human+adipose+stromal+cells+for+clinical+applications.">Use of freshly isolated human adipose stromal cells for clinical applications.</a> Aesthetic Surgery Journal. 37, S4-S8 (2017).</li><li>Morita, S., Kojima, T., Kitamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plat-E:+an+efficient+and+stable+system+for+transient+packaging+of+retroviruses.">Plat-E: an efficient and stable system for transient packaging of retroviruses.</a> Gene Therapy. 7 (12), 1063-1066 (2000).</li><li>Li, Y., Fromme, T., Klingenspor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meaningful+respirometric+measurements+of+UCP1-mediated+thermogenesis.">Meaningful respirometric measurements of UCP1-mediated thermogenesis.</a> Biochimie. 134, 56-61 (2017).</li><li>Hiraike, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+immunoprecipitation+with+mouse+adipocytes+using+hypotonic+buffer+to+enrich+nuclear+fraction+before+fixation.">Chromatin immunoprecipitation with mouse adipocytes using hypotonic buffer to enrich nuclear fraction before fixation.</a> STAR Protocols. 4 (1), 102093 (2023).</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></ol>

None of the authors have a competing financial interest related to this work.

Here, we described a protocol for isolation of the SVF from murine adipose tissue to obtain preadipocytes and perform adipocyte differentiation in vitro. The use of a tissue dissociator for collagenase digestion decreased experimental variation, decreased the risk of contamination, and increased reproducibility. While this procedure is a critical step within the presented protocol, the process is highly automated and optimization is not needed. However, depending on the mouse age and adipose tissue depot, optimi...

Adipose tissue biology has been attracting ever-increasing attention because of the growing prevalence of obesity and type 2 diabetes globally1. Adipocytes store excess energy in the form of lipid droplets, which are released upon starvation. Moreover, adipose tissue maintains systemic energy homeostasis by serving as an endocrine organ and communicating with other tissues2,3. Intriguingly, both excess adipose tissue (obesity) and adipose loss (lipodystrophy) are linked to insulin resistance and diabetes1. Adipocytes are divided into three types: white, brown, and beige1. White adipocytes mainly store excess energy as lipids, whereas brown and beige adipocytes dissipate energy in the form of heat via mitochondrial uncoupling protein-1 (Ucp1)1,4. Notably, beige adipocytes (also called &#34;inducible&#34; brown adipocytes) appear in white adipose tissue in response to cold or sympathetic stimulation and exhibit gene expression patterns that overlap with but are distinct from those of &#34;classical&#34; brown adipocytes5. Recently, brown and beige adipocytes have been anticipated as potential targets of anti-obesity and anti-diabetes treatments aimed at &#34;enhancing energy dissipation&#34; rather than &#34;suppressing energy intake&#34;4. Supportively, the risk allele of the FTO obesity variant rs1421085 in humans, which exhibits the strongest association with higher body mass index (BMI) among common variants6,7 and exhibits various gene-environment interactions8,9, is reported to negatively regulate beige adipocyte differentiation and function10. Peroxisome proliferator-activated receptor &#947; (PPAR&#947;) is known as a master transcriptional regulator of adipogenesis and is necessary and sufficient for adipocyte differentiation11. Transcriptional regulators, such as PRD1-BF1-RIZ1 homologous domain containing 16 (PRDM16), early b cell factor 2 (EBF2), and nuclear factor I-A (NFIA), are crucial for brown and beige adipocyte differentiation and function12,13,14,15,16,17,18. On the other hand, white adipocyte gene programming requires transcriptional regulators, such as transducin-like enhancer protein 3 (TLE3) and zinc finger protein 423 (ZFP423)19,20,21.
In vitro model systems enable molecular studies to be performed that aim to improve the understanding of the mechanism(s) underlying the functions and dysfunctions of adipocytes. Although publicly available and immortalized preadipocyte cell lines such as 3T3-L1 and 3T3-F442A exist22,23,24, the culture of primary preadipocytes and differentiation into adipocytes would be a more suitable model for studying in vivo adipogenesis. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is a well-known method for obtaining primary preadipocytes25,26. However, collagenase digestion of adipose tissue, which is commonly performed using a bacterial shaker with a tube rack, can result in experimental variation and is prone to contamination27,28. Here, we describe an alternative protocol that uses a gentle magnetic-activated cell sorting (MACS) tissue dissociator for collagenase digestion to achieve easier isolation of the SVF.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mm dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>12 well plate</td>
			<td>Corning</td>
			<td>3513</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm dish</td>
			<td>IWAKI</td>
			<td>3010-060</td>
			<td></td>
		</tr>
		<tr>
			<td>Adipose Tissue Dissociation Kit, mouse and rat</td>
			<td>Miltenyi Biotec</td>
			<td>130-105-808</td>
			<td>contents: Enzyme D, Enzyme R, Enzyme A and Buffer A</td>
		</tr>
		<tr>
			<td>Cell strainer 70 &#181;m</td>
			<td>BD falcon</td>
			<td>#352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen coated dishes, 100 mm</td>
			<td>BD</td>
			<td>#356450</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen coated dishes, 60 mm</td>
			<td>BD</td>
			<td>#354401</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen I Coat Microplate 6 well</td>
			<td>IWAKI</td>
			<td>4810-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Wako</td>
			<td>041-18861</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Forceps</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>autoclave before use</td>
		</tr>
		<tr>
			<td>Dissecting Scissors, blunt/sharp</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>autoclave before use</td>
		</tr>
		<tr>
			<td>Dissecting Scissors, sharp/sharp</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>autoclave before use</td>
		</tr>
		<tr>
			<td>DMEM/F-12, GlutaMAX supplement</td>
			<td>Gibco</td>
			<td>10565-042</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS C Tubes</td>
			<td>Milteny Biotec</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACSOcto Dissociator with Heaters</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-427</td>
			<td></td>
		</tr>
		<tr>
			<td>Humulin R Injection U-100</td>
			<td>Eli Lilly</td>
			<td>872492</td>
			<td></td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Sigma</td>
			<td>I7378-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Isobutylmethylxanthine (IBMX)</td>
			<td>Sigma</td>
			<td>17018-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Life Technologies</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Neomycin Sulfate</td>
			<td>Fujifilm</td>
			<td>146-08871&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Invitrogen&#160;</td>
			<td>31985-062</td>
			<td></td>
		</tr>
		<tr>
			<td>pBABE-neo largeTcDNA (SV40)</td>
			<td>Add gene</td>
			<td>#1780</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS tablets</td>
			<td>Takara</td>
			<td>T900</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum-E (Plat-E) Retroviral Packaging Cell Line</td>
			<td>cell biolab</td>
			<td>RV-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Nacalai Tesque</td>
			<td>12996-81</td>
			<td></td>
		</tr>
		<tr>
			<td>Power Sybr Green Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4367659</td>
			<td></td>
		</tr>
		<tr>
			<td>ReverTra Ace qPCR RT Master Mix</td>
			<td>TOYOBO</td>
			<td>#FSQ-201</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit (250)</td>
			<td>QIAGEN</td>
			<td>74106</td>
			<td></td>
		</tr>
		<tr>
			<td>Rosiglitazone</td>
			<td>Wako</td>
			<td>180-02653</td>
			<td></td>
		</tr>
		<tr>
			<td>T3</td>
			<td>Sigma</td>
			<td>T2877-100mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%)</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
	</tbody>
</table>

semi-automated isolation of the stromal vascular fraction from murine white adipose tissue using a tissue dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

Author Spotlight: Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator  

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

We are interested in brown and beige subtypes of adipocyte which dissipate energy in the form of heat, while white adipocyte generally store energy as Our goal is to develop a new treatment for obesity and diabetes based on promotion of energy expenditure, rather than suppression of energy intake. We have identified a transcription factor, NFI, as a critical regulator of brown adipocyte differentiation by a genome-wide open chromatin analysis. NFI and the master transcriptional regulator of adipogenesis, PPAR-gamma, co-localize as the brown-fat-specific enhancers.In the process of isolating stromal vascular fraction from mouse adipose tissue, manual mincing and collagenase digestion of adipose tissue can lead to experimental variability and contamination. Our method uses a tissue dissociator for collagenase digestion, which facilitates SVF isolation, reduce experimental variability and contamination, and improves reproducibility. Experiment using individually-differentiated adipocyte are indispensable for research in our field.Our protocol would offer easy and reproducible SVF isolation for subsequent adipocyte differentiation and would accelerate research in our community. Currently, we are investigating the role of NFI of all-body metabolism, such as body weight and glucose, using gain and loss of function mouse models. We will also explore other pharmacological activation of NFI for each downstream pathway in near future.

This protocol yields fully differentiated, lipid-laden adipocytes 7 days after inducing adipocyte differentiation. The degree of adipocyte differentiation can be evaluated by the oil red o staining of triglycerides and lipids (Figure 1A), or mRNA expression analysis using qPCR-RT of adipocyte genes, such as the master regulator of adipogenesis Pparg and its target Fabp4 (Figure 1B). To induce beige adipocyte differentiation in vitro, a...

Watch this Scientific Journal Video about Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator at JoVE.com

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

semi-automated-isolation-stromal-vascular-fraction-from-murine-white

Research

JoVE Journal

Biology

Isolation, Digestion, Filtration and Cell Plating of Murine Adipose Tissue

Begin by placing a seven to eight week old euthanized male mouse on a clean bench surface. Cut the abdominal skin of the mouse with a pair of scissors towards the lower abdomen. Excise the adipose tissue from the inner thighs.Place the excised tissue into tube C on ice containing 2.5 milliliters of an enzyme mixture of enzymes D, R, A and 2.35 milliliters of DMEM/F12 without FBS or antibiotics. Using scissors, cut the adipose tissue into approximately two square millimeter sized pieces. Tightly close the cap of tube C and invert the tube.Attach the tube to the sleeve of a tissue dissociator and digest the samples at 37 degrees Celsius for 40 minutes. Next, prewarm DMEM/F12 media containing FBS and antibiotics to 37 degrees Celsius. After incubation, remove tube C from the tissue dissociator and stop digestion by adding five milliliters of DMEM/F12 with FBS and antibiotics.Gently pipette four times. Centrifuge the suspension at 700 g for 10 minutes at 20 degrees Celsius. Without disturbing the cell palette, carefully aspirate the supernatant.Resuspend the pellet in 10 milliliters of DMEM/F12 containing FBS and antibiotics, and gently pipette five times. Filter the cell suspension through a 70 micron diameter cell strainer placed on a 50 milliliter tube. Centrifuge the filtrate at 250 g for five minutes.Resuspend the pellet in 10 milliliters of PBS. Before plating, centrifuge the cell suspension at 500 g for five minutes. Discard the supernatant.Resuspend the pellet in 10 milliliters of DMEM/F12 containing FBS and antibiotics. Mix by pipetting the suspension gently, 10 times. Plate 10 milliliters of the suspension on a 10 centimeter collagen coated dish.Place the dish in a cell culture incubator. Aspirate the medium and wash the cells three times with three milliliters of PBS per wash. Add 10 milliliters of DMEM/F12 containing FBS and antibiotics and incubate.Oil Red O staining showed lipid laden adipocytes seven days after inducing adipocyte differentiation. The degree of full differentiation was confirmed by mRNA expression analysis of adipogenesis regulators. Rosiglitazone induced a dose dependent effect on the expression levels of brown fat specific genes such as Ucp1 and Ppargc1a.However, for Fabp4, the effect saturated at 0.1 micromole rosiglitazone concentration. Differentiated adipocytes can be used for various functional and mechanistic analyses such as oxygen consumption rate analysis and chromatin immunoprecipitation.

1.5K Views.  The University of Tokyo.  This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

Semi-Automated Isolation of Murine White Adipose Tissue

Summary