Funding was provided by FONDECYT (1181214 and 1221146) and Anillos (ACT210039) to VC and doctoral scholarships ANID 21171743 to AMF and ANID 21150665 to FS. We thank Alejandro Munizaga for help in the processing of the samples and technical advice for the transmission electron microscopy. The illustrations were produced using BioRender.

The animal procedures were approved by the Institutional Animal Care and Use Committee at Pontificia Universidad Cat&#243;lica de Chile. P0.5 newborn mice of both sexes, derived from a mixed background of C57BL/6J and 129J strains, were used for this study.
1.&#160;Tissue extraction
<ol>
	<li>Clean and disinfect the workbench with a 70% ethanol solution, and use sterile surgical material.</li>
	<li>Euthanize P0.5 newborn pups by decapitation with surgical scissors following institutionally approved protocol.</li>
	<li>Place the mouse in a prone position (facing downward), and make a 1 cm long incision in the skin at the mid-level of the animal&#39;s back using sharp scissors. Remove the skin carefully. 
	NOTE: This step is critical because if the skin is not well removed, it might tear and damage the iBAT and preclude the optimal preparation of the SVF.</li>
	<li>Surgically detach the iBAT from the carcass using small scissors. 
	&#8203;NOTE: The iBAT is visualized as two fatty lobules that can be removed independently.</li>
	<li>Harvest both iBAT lobes by using curved-tip pliers to remove the lobes independently.</li>
	<li>Place the two lobes in a plastic tube with 1.5 mL of sterile PBS at room temperature. Maintain the tissues in their individual tubes until the tissue extraction is completed for all the pups required.</li>
</ol>
2. Tissue digestion
<ol>
	<li>Aliquot 300 &#181;L of collagenase type II digestion buffer (0.2% type II collagenase in buffer [25 mM KHCO3, 1.2 mM MgSO4, 120 mM NaCl, 5 mM glucose, 12 mM KH2PO4, 4.8 mM KCl, 1.2 mM CaCl2, 2.5% BSA, and 1% penicillin/streptomycin, at pH 7.4]) (Table 1) in 1.5 mL tubes in a biosafety cabinet. 
	NOTE: Prepare sufficient tubes for the number of individual tissues to be extracted. In the present study, seven tubes were used, corresponding to a litter of seven pups.</li>
	<li>Move to the bench, and place the iBAT lobes in collagenase type II digestion buffer to degrade the extracellular matrix.</li>
	<li>Incubate the iBAT tissues for 45 min at 37 &#176;C with continuous gentle shaking at 800 rpm (Figure 1).</li>
</ol>
3. Tissue processing
NOTE: After the digestion, perform all the following steps in a class II laminar flow tissue culture hood.
<ol>
	<li>Prepare and label two sets of 1.5 mL plastic tubes for each individual sample.</li>
	<li>Mechanically disrupt the tissue suspension in collagenase type II digestion buffer by pipetting up and down using a P1,000 micropipette and filtered tips. Use a new tip for each sample. 
	NOTE: This is a critical step. Ensure to mechanically disaggregate the tissue with the tip and pipette up and down until macroscopic homogenization is complete.</li>
	<li>Pass the disaggregated tissues through individual 100 &#181;m cell strainers (see the Table of Materials) into new 1.5 mL tubes.</li>
	<li>Add 500 &#181;L of ACK buffer (see the Table of Materials) to the filtered tissue samples, invert the tubes 4-5 times, and incubate at room temperature for 4 min. 
	NOTE: Do not exceed the incubation time, as this may damage the cells of interest.</li>
	<li>Centrifuge the tissue samples at 300 x g for 5 min at room temperature.</li>
	<li>Discard the supernatant by inverting the tube, and resuspend the pellet in 1 mL of culture medium (DMEM-F12 with 10% FBS, 1% antibiotic antimycotic, pH 7.2) (see the Table of Materials).</li>
	<li>Pass the suspensions through a 40 &#181;m cell strainer into new 1.5 mL tubes using a P1,000 micropipette and filtered tips.</li>
	<li>Centrifuge the samples at 300 x g for 5 min at room temperature. 
	NOTE: The pellet corresponds to the preadipocyte-containing SVF.</li>
	<li>Discard the supernatant by inverting the tube, and resuspend the pellet in 1 mL of culture medium by pipetting.</li>
	<li>Prepare one 15 mL tube for each sample by adding 5 mL of culture medium.</li>
	<li>Add each resuspended pellet to its corresponding 15 mL tube containing 5 mL of culture medium. Invert the tube 4-5 times to mix the cell content.</li>
	<li>Seed each sample into six wells of a 24-well culture plate by adding 1 mL of the suspension to each well (Figure 1).</li>
</ol>
4. Stromal vascular fraction (SVF) culture
<ol>
	<li>Change the culture medium every other day until the cells reach 100% confluence. 
	NOTE: During this period, non-plastic adherent cells are removed, resulting in a primary culture enriched with a preadipocyte-containing SVF.</li>
	<li>When the cells become 100% confluent, remove the culture medium, and wash the cells with 800 &#181;L of sterile PBS.</li>
	<li>Add 150 &#181;L of 0.25% trypsin-EDTA (see the Table of Materials) to every well, and place the culture plate for 3 min in the incubator for the detachment of the cells. 
	NOTE: Verify that the cells are detached using a phase contrast light microscope.</li>
	<li>Inactivate the trypsin by adding 850 &#181;L of culture medium to each well.</li>
	<li>Collect the cells from the six wells seeded in step 3.12, and transfer them into a 15 mL tube.</li>
	<li>Make the total volume up to 12 mL by adding 6 mL of culture medium. Invert the tube 4-5 times to mix the content.</li>
	<li>Seed 1 mL of the sample into each well of a 12-well plate. Change the culture medium every other day until the cells reach confluence.</li>
	<li>When the cells become 100% confluent, detach them with 150 &#181;L of 0.25% trypsin-EDTA following the same incubation conditions as in step 4.3, and seed them according to the surface area of the well required for the experiment.</li>
	<li>Perform high-resolution imaging after fixing the cells following step 8. 
	NOTE: A density of 35,000 cells/cm&#178; is recommended for further experiments (protein and total RNA extraction, 330,000 cells [one well of a 6-well plate]; immunofluorescence [IF], 10,500 cells [one well of a 96-well plate]).</li>
</ol>
5. Adipogenic differentiation
<ol>
	<li>Incubate the cells with induction medium (DMEM-F12 with 10% FBS, 1% antibiotic antimycotic, pH 7.2, supplemented with 500 nM dexamethasone, 125 nM indomethacin, 0.5 mM 3-isobutyl-1-methylxanthine [IBMX], 5 &#181;M rosiglitazone, 1 nM T3, and 20 nM insulin) (Table 1) for 72 h (day 0 to day 2) (Figure 2). 
	NOTE: The culture medium must be changed every other day; thus, the first medium change must occur on day 2 after the initial induction.</li>
	<li>Change to maintenance medium (DMEM-F12 with 10% FBS, 1% antibiotic antimycotic, pH 7.2, supplemented with 5 &#181;M rosiglitazone, 1 nM T3, and 20 nM insulin) (Table 1) at day 3, and maintain this maintenance medium until day 7 (Figure 2). 
	&#8203;NOTE: The maintenance medium must be changed on day 5 and day 7. Small lipid droplets can be visualized by phase contrast microscopy from day 3 of differentiation. Large lipid droplets are evident on day 7 of differentiation. Differentiation procedures longer than 7 days result in cell loss by detachment, likely due to the high buoyancy of lipid-laden cells.</li>
</ol>
6. Cellular homogenate preparation
<ol>
	<li>Remove the culture medium at day 0 and day 7 of differentiation, and wash the cells 3 times with sterile PBS.</li>
	<li>After the last cleaning, add 1.3 mL of sterile PBS to each well. 
	NOTE: Maintain the cells in ice until the cleaning of the last plate.</li>
	<li>Harvest the cells mechanically from one well of a 6-well plate (~330,000 cells) with a cell scraper, and put the cellular suspension in a 1.5 mL tube.</li>
	<li>Centrifuge the samples at 2,800 x g for 5 min at 4 &#176;C.</li>
	<li>Remove the PBS with a micropipette, and maintain the tubes on ice.</li>
	<li>Resuspend the cellular pellet in RIPA buffer with protease and phosphatase inhibitors (see the Table of Materials), and incubate for 30 min on ice. 
	NOTE: To lysate the cellular pellet, 70-80 &#181;L are sufficient.</li>
	<li>Centrifuge the samples at 21,000 x g for 15 min at 4 &#176;C.</li>
	<li>Carefully remove the supernatant to a 1.5 mL tube, and discard the pellet. 
	NOTE: The supernatant represents the cellular homogenate.</li>
	<li>Determine the protein concentration of the cellular homogenate using bicinchoninic acid (BCA) for the colorimetric detection and quantitation of the total protein18,19.</li>
</ol>
7. Western blotting
<ol>
	<li>Prepare 30 &#181;g of the cellular homogenate (from step 6.8) and add commercially available SDS-PAGE sample loading buffer (see the Table of Materials).</li>
	<li>Resolve in polyacrylamide gel electrophoresis-SDS20 (PAGE-SDS) according to the molecular weight of every protein. 
	NOTE: Use 10% PAGE-SDS for PPAR&#947;, C/EBP&#945;, PLIN1, CD36, and UCP1, 12% PAGE-SDS for OXPHOS, and 15% PAGE-SDS for TOM20 (see the Table of Materials).</li>
	<li>Electrotransfer the proteins onto the PVDF membrane (see the Table of Materials) at 350 mA for 2 h. 
	NOTE: The ice container must be changed after 1 h. During the entire process, maintain the chamber on ice.</li>
	<li>Block the membranes with 5% BSA or fat-free milk in Tris-buffered saline (TBS) 0.1% Tween 20 (TBS-T) for 2 h following the recommendations from the antibodies&#39; datasheets.</li>
	<li>Incubate with the primary antibodies (the antibodies used in this study are detailed in the Table of Materials) overnight at 4 &#176;C. 
	NOTE: All the antibodies used in this study were diluted to 1:1,000 in blocking solution.</li>
	<li>Wash the membranes 3 times with 0.1% TBS-T, and incubate them with horseradish peroxidase (HRP) conjugated secondary antibody (see the Table of Materials) for 2 h at room temperature.</li>
	<li>Wash the membranes 3 times with 0.1%. TBS-T. Perform one last wash with 1x TBS. Store the membranes in 1x TBS until visualization. Storing for a maximum of 1 h is recommended.</li>
	<li>Visualize the membranes by chemiluminescence20 or any other preferred method.</li>
</ol>
8. High-resolution imaging
<ol>
	<li>Perform the immunofluorescence assay following the steps below.
	<ol>
		<li>Seed and differentiate the primary cultures of brown preadipocytes from P0.5 newborn mice (following step 4.8) in 96-well plates with an optical bottom (see the Table of Materials).</li>
		<li>Fix the cells at day 0 and day 7 of differentiation with 4% PFA for 20 min, and wash 3 times with sterile PBS.</li>
		<li>Permeabilize the cells with 0.1% Triton X-100 for 15 min, and block them with 3% fish gelatin (see the Table of Materials) for 1 h at room temperature.</li>
		<li>Wash 3 times with sterile PBS, and incubate with PLIN1 antibody (1:400, see the Table of Materials) in 1% BSA/PBS overnight at 4 &#176;C in a humid chamber.</li>
		<li>Wash 3 times with sterile PBS, and incubate with fluorophore-conjugated secondary antibody at 1:1,000 (1% BSA/PBS) (see the Table of Materials) for 1 h at room temperature.</li>
		<li>Stain the nuclei with 1 &#181;g/mL Hoechst 33342 and the lipid droplets with 1 &#181;g/mL BODIPY for 30 min at room temperature. 
		NOTE: The fluorescence images were acquired using a cell imaging system (see the Table of Materials). Overall, 9 or 16 photos per well were acquired with two to three channels with a magnification of 20x.</li>
	</ol>
	</li>
	<li>Perform transmission electron microscopy following the steps below.
	<ol>
		<li>Grow and differentiate the brown preadipocytes in 35 mm x 10 mm cell culture dishes.</li>
		<li>On day 0 and day 7 of differentiation, remove the medium, and fix the cells for 1 h with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.0 (see the Table of Materials).</li>
		<li>Wash the cells 3 times with 0.05 M sodium cacodylate buffer for 10 min, and treat them with 1% aqueous osmium tetroxide (see the Table of Materials) for 15 min.</li>
		<li>Wash the cells 3 times with bi-distilled water for 5 min, and treat them with 1% aqueous uranyl acetate (see the Table of Materials) for 15 min.</li>
		<li>Dehydrate the cells with an ethanol gradient of 30%, 50%, 70%, 95%, and 100% for 5 min each time.</li>
		<li>Pre-embed the cells in 1:1 epon/ethanol for 30 min and then in epon for 1 h (see the Table of Materials). 
		NOTE: The inclusion of cells is performed directly in the dish by adding fresh epon to cover the entire cell culture monolayer. Let the epon polymerize in an oven at 60 &#176;C for 24 h.</li>
		<li>Immerse the cell culture dish in liquid nitrogen for 2 min, and then fracture it using pliers to loosen the plastic inclusion.</li>
		<li>Glue the inclusion segments with cells to a block of resin to obtain fine cuts (80 nm) parallel to the surface in an ultramicrotome (see the Table of Materials).</li>
		<li>Stain the cuts with 1% aqueous uranyl acetate for 4 min and lead citrate for 5 min, according to Reynolds et al.21.</li>
		<li>Visualize the grids in an electron microscope (see the Table of Materials).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Luo, L., Liu, 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+tissue+in+control+of+metabolism.">Adipose tissue in control of metabolism.</a> Journal of Endocrinology. 231 (3), 77-99 (2016).</li><li>Gaspar, R. C., Pauli, J. R., Shulman, G. I., Mu&#241;oz, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+update+on+brown+adipose+tissue+biology:+A+discussion+of+recent+findings.">An update on brown adipose tissue biology: A discussion of recent findings.</a> American Journal of Physiology-Endocrinology and Metabolism. 320 (3), 488-495 (2021).</li><li>Richard, D., Picard, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+fat+biology+and+thermogenesis.">Brown fat biology and thermogenesis.</a> Frontiers in Bioscience. 16, 1233-1260 (2011).</li><li>Rosenwald, M., Wolfrum, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+definition+of+brite+versus+white+and+classical+brown+adipocytes.">The origin and definition of brite versus white and classical brown adipocytes.</a> Adipocyte. 3 (1), 4-9 (2014).</li><li>Smorlesi, A., Frontini, A., Giordano, A., Cinti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adipose+organ:+White-brown+adipocyte+plasticity+and+metabolic+inflammation.">The adipose organ: White-brown adipocyte plasticity and metabolic inflammation.</a> Obesity Reviews. 13, 83-96 (2012).</li><li>Townsend, K., Tseng, Y. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+adipose+tissue:+Recent+insights+into+development,+metabolic+function+and+therapeutic+potential.">Brown adipose tissue: Recent insights into development, metabolic function and therapeutic potential.</a> Adipocyte. 1 (1), 13-24 (2012).</li><li>Benador, I. 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=Mitochondria+bound+to+lipid+droplets+have+unique+bioenergetics,+composition,+and+dynamics+that+support+lipid+droplet+expansion.">Mitochondria bound to lipid droplets have unique bioenergetics, composition, and dynamics that support lipid droplet expansion.</a> Cell Metabolism. 27 (4), 869-885 (2018).</li><li>Benador, I. Y., Veliova, M., Liesa, M., Shirihai, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+bound+to+lipid+droplets:+Where+mitochondrial+dynamics+regulate+lipid+storage+and+utilization.">Mitochondria bound to lipid droplets: Where mitochondrial dynamics regulate lipid storage and utilization.</a> Cell Metabolism. 29 (4), 827-835 (2019).</li><li>Lee, P., Swarbrick, M. M., Ho, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+adipose+tissue+in+adult+humans:+A+metabolic+renaissance.">Brown adipose tissue in adult humans: A metabolic renaissance.</a> Endocrine Reviews. 34 (3), 413-438 (2013).</li><li>Han, S., Sun, H. M., Hwang, K. C., Kim, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stromal+vascular+fraction+cells:+Update+on+clinical+utility+and+efficacy.">Adipose-derived stromal vascular fraction cells: Update on clinical utility and efficacy.</a> Critical Reviews in Eukaryotic Gene Expression. 25 (2), 145-152 (2015).</li><li>Klein, 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=Beta(3)-adrenergic+stimulation+differentially+inhibits+insulin+signaling+and+decreases+insulin-induced+glucose+uptake+in+brown+adipocytes.">Beta(3)-adrenergic stimulation differentially inhibits insulin signaling and decreases insulin-induced glucose uptake in brown adipocytes.</a> Journal of Biological Chemistry. 274 (3), 34795-34802 (1999).</li><li>Gaom, W., Kong, X., Yang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+primary+culture,+and+differentiation+of+preadipocytes+from+mouse+brown+adipose+tissue.">Isolation, primary culture, and differentiation of preadipocytes from mouse brown adipose tissue.</a> Methods in Molecular Biology. 1566, 3-8 (2017).</li><li>Rocha, A. L., Guerra, B. A., Boucher, J., Mori, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+to+induce+brown/beige+adipocyte+differentiation+from+murine+preadipocytes.">A Method to induce brown/beige adipocyte differentiation from murine preadipocytes.</a> Bio-protocol. 11 (24), 4265 (2021).</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>Tapia, P. 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=Absence+of+AGPAT2+impairs+brown+adipogenesis,+increases+IFN+stimulated+gene+expression+and+alters+mitochondrial+morphology.">Absence of AGPAT2 impairs brown adipogenesis, increases IFN stimulated gene expression and alters mitochondrial morphology.</a> Metabolism. 111, 154341 (2020).</li><li>Gonz&#225;lez-H&#243;dar, 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=Decreased+caveolae+in+AGPAT2+lacking+adipocytes+is+independent+of+changes+in+cholesterol+or+sphingolipid+levels:+A+whole+cell+and+plasma+membrane+lipidomic+analysis+of+adipogenesis.">Decreased caveolae in AGPAT2 lacking adipocytes is independent of changes in cholesterol or sphingolipid levels: A whole cell and plasma membrane lipidomic analysis of adipogenesis.</a> Biochimica et Biophysica Acta - Molecular Basis of Disease. 1867 (9), 166167 (2021).</li><li>Fern&#225;ndez-Galilea, M., Tapia, P., Cautivo, K., Morselli, E., Cort&#233;s, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AGPAT2+deficiency+impairs+adipogenic+differentiation+in+primary+cultured+preadipocytes+in+a+non-autophagy+or+apoptosis+dependent+mechanism.">AGPAT2 deficiency impairs adipogenic differentiation in primary cultured preadipocytes in a non-autophagy or apoptosis dependent mechanism.</a> Biochemical and Biophysical Research Communications. 467 (1), 39-45 (2015).</li><li>Walker, 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=The+bicinchoninic+acid+(BCA)+assay+for+protein+quantitation.">The bicinchoninic acid (BCA) assay for protein quantitation.</a> The Protein Protocols Handbook. , 11-15 (2009).</li><li>Smith, P. 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=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Analytical Biochemistry. 150 (1), 76-85 (1985).</li><li>Kurien, B. T., Scofield, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting.">Western blotting.</a> Methods. 38 (4), 283-293 (2006).</li><li>Reynolds, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+lead+citrate+at+high+pH+as+an+electron-opaque+stain+in+electron+microscopy.">The use of lead citrate at high pH as an electron-opaque stain in electron microscopy.</a> Journal of Cell Biology. 17 (1), 208-212 (1963).</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>Ambele, M. A., Dhanraj, P., Giles, R., Pepper, 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=Adipogenesis:+A+complex+interplay+of+multiple+molecular+determinants+and+pathways.">Adipogenesis: A complex interplay of multiple molecular determinants and pathways.</a> International Journal of Molecular Sciences. 21 (12), 4283 (2020).</li><li>Lefterova, M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PPAR&#947;+and+C/EBP+factors+orchestrate+adipocyte+biology+via+adjacent+binding+on+a+genome-wide+scale.">PPAR&#947; and C/EBP factors orchestrate adipocyte biology via adjacent binding on a genome-wide scale.</a> Genes &amp; Development. 22 (21), 2941-2952 (2008).</li><li>Ntambi, J. M., Young-Cheul, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipocyte+differentiation+and+gene+expression.">Adipocyte differentiation and gene expression.</a> The Journal of Nutrition. 130 (12), (2000).</li><li>Itabe, H., Yamaguchi, T., Nimura, S., Sasabe, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perilipins:+A+diversity+of+intracellular+lipid+droplet+proteins.">Perilipins: A diversity of intracellular lipid droplet proteins.</a> Lipids in Health and Disease. 16, 83 (2017).</li><li>Christiaens, V., Van Hul, M., Lijnen, H. R., Scroyen, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD36+promotes+adipocyte+differentiation+and+adipogenesis.">CD36 promotes adipocyte differentiation and adipogenesis.</a> Biochimica et Biophysica Acta - General Subjects. 1820 (7), 949-956 (2012).</li><li>Carobbio, S., Rosen, B., Vidal-Puig, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipogenesis:+New+insights+into+brown+adipose+tissue+differentiation.">Adipogenesis: New insights into brown adipose tissue differentiation.</a> Journal of Molecular Endocrinology. 51 (3), 75-85 (2013).</li><li>Ricquier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncoupling+protein+1+of+brown+adipocytes,+the+only+uncoupler:+A+historical+perspective.">Uncoupling protein 1 of brown adipocytes, the only uncoupler: A historical perspective.</a> Frontiers in Endocrinology. 2, 85-91 (2011).</li><li>Cui, L., Mirza, A. H., Zhang, S., Liang, B., Liu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+droplets+and+mitochondria+are+anchored+during+brown+adipocyte+differentiation.">Lipid droplets and mitochondria are anchored during brown adipocyte differentiation.</a> Protein &amp; Cell. 10 (12), 921-926 (2019).</li><li>Hao, 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=Indomethacin+enhances+brown+fat+activity.">Indomethacin enhances brown fat activity.</a> Journal of Pharmacology and Experimental Therapeutics. 365 (3), 467-475 (2018).</li><li>Lehmann, J. M., Lenhard, J. M., Oliver, B. B., Ringold, G. M., Kliewer, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisome+proliferator-activated+receptors+alpha+and+gamma+are+activated+by+indomethacin+and+other+non-steroidal+anti-inflammatory+drugs.">Peroxisome proliferator-activated receptors alpha and gamma are activated by indomethacin and other non-steroidal anti-inflammatory drugs.</a> Journal of Biological Chemistry. 272 (6), 3406-3410 (1997).</li><li>Scott, M. A., Nguyen, V. T., Levi, B., James, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+methods+of+adipogenic+differentiation+of+mesenchymal+stem+cells.">Current methods of adipogenic differentiation of mesenchymal stem cells.</a> Stem Cells and Development. 20 (10), 1793-1804 (2011).</li><li>Wilson-Fritch, 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=Mitochondrial+remodeling+in+adipose+tissue+associated+with+obesity+and+treatment+with+rosiglitazone.">Mitochondrial remodeling in adipose tissue associated with obesity and treatment with rosiglitazone.</a> The Journal of Clinical Investigation. 114 (9), 1281-1289 (2004).</li><li>Cannon, B., Nedergaard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+adipose+tissue:+Function+and+physiological+significance.">Brown adipose tissue: Function and physiological significance.</a> Physiological Reviews. 84 (1), 277-359 (2004).</li><li>Ohno, H., Shinoda, K., Spiegelman, B. M., 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=PPARgamma+agonists+induce+a+white-to-brown+fat+conversion+through+stabilization+of+PRDM16+protein.">PPARgamma agonists induce a white-to-brown fat conversion through stabilization of PRDM16 protein.</a> Cell Metabolism. 15 (3), 395-404 (2012).</li><li>Merlin, 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=Rosiglitazone+and+a+&#946;3-adrenoceptor+agonist+are+both+required+for+functional+browning+of+white+adipocytes+in+culture.">Rosiglitazone and a &#946;3-adrenoceptor agonist are both required for functional browning of white adipocytes in culture.</a> Frontiers in Endocrinology. 9, 249 (2018).</li><li>Fayyad, A. 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=Rosiglitazone+enhances+browning+adipocytes+in+association+with+MAPK+and+PI3-K+pathways+during+the+differentiation+of+telomerase-transformed+mesenchymal+stromal+cells+into+adipocytes.">Rosiglitazone enhances browning adipocytes in association with MAPK and PI3-K pathways during the differentiation of telomerase-transformed mesenchymal stromal cells into adipocytes.</a> International Journal of Molecular Sciences. 20 (7), 1618-1633 (2019).</li><li>Zennaro, M. -. 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=Hibernoma+development+in+transgenic+mice+identifies+brown+adipose+tissue+as+a+novel+target+of+aldosterone+action.">Hibernoma development in transgenic mice identifies brown adipose tissue as a novel target of aldosterone action.</a> The Journal of Clinical Investigation. 101 (6), 1254-1260 (1998).</li><li>Klaus, 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=Characterization+of+the+novel+brown+adipocyte+cell+line+HIB+1B.+Adrenergic+pathways+involved+in+regulation+of+uncoupling+protein+gene+expression.">Characterization of the novel brown adipocyte cell line HIB 1B. Adrenergic pathways involved in regulation of uncoupling protein gene expression.</a> Journal of Cell Science. 107 (1), 313-319 (1994).</li><li>Guennoun, 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=Comprehensive+molecular+characterization+of+human+adipocytes+reveals+a+transient+brown+phenotype.">Comprehensive molecular characterization of human adipocytes reveals a transient brown phenotype.</a> Journal of Translational Medicine. 13, 135 (2015).</li></ol>

The authors have nothing to disclose.

The present protocol is a simple and replicable two-phase differentiation procedure (Figure 2) for generating cells with the molecular and morphological characteristics of mature brown adipocytes. The surgical harvesting of the iBAT is the first critical step because tissue tearing severely limits the viability of the starting material. Tissue processing is also key because a homogeneous cell suspension that is free of debris greatly increases the amount of SVF that can be cultured. In the c...

White and brown adipose tissue differ in their anatomical location, cellular origin, function, morphology, and total mass. White adipose tissue (WAT) is the major physiological energy reservoir of the body and stores large amounts of triacylglycerol (TAG) in highly specialized cells that have a single giant lipid droplet occupying most of their cellular volume1. TAG lipolysis releases free fatty acids, which enter the systemic circulation to meet energy demands during fasting or other states of negative energy balance. Additionally, the WAT secretes protein and lipid products, called adipokines and lipokines, respectively, that have metabolic, immune, and reproductive regulatory functions, thus making the WAT the largest endocrine tissue in the body2.
Brown adipose tissue (BAT) is a much smaller organ whose main physiological function is non-shivering thermogenesis to prevent hypothermia. In mice and newborn humans, BAT is a well-defined organ located in the interscapular space. Adult humans lack interscapular BAT (iBAT); nevertheless, they develop clusters of brown adipocyte-like cells integrated in depots that otherwise mostly comprise WAT. These &#34;brown-in-white&#34; (brite) adipocytes share morphological and molecular features with classical iBAT adipocytes, but they have a different cellular origin3,4.
In contrast to white adipocytes, brown adipocytes have multiple small lipid droplets and abundant mitochondria5. Uncoupling protein 1 (UCP1, also known as thermogenin) is uniquely expressed by brown and brite adipocytes and mediates proton leakage in the inner mitochondrial membrane (IMM), thus uncoupling electron transport from ATP synthesis and generating heat. Non-shivering thermogenesis in BAT is activated by norepinephrine (NE), which is released from the sympathetic terminals in the BAT in response to cold stimulation6. NE binds to beta-adrenoceptors (mostly beta 3) on the surface of brown adipocytes and triggers an intracellular cAMP-mediated signaling cascade. This results in TAG lipolysis, the beta-oxidation of mitochondrial fatty acids, and heat generation upon UCP1 activation3. The close functional relationship between lipid droplets and mitochondria in brown adipocytes has structural parallels, such as the interaction between these organelles in areas that are large and have very tight physical contact7,8.
iBAT has abundant blood vessels and sympathetic terminals9. These structures, along with the preadipocytes, immune cells, fibroblasts, and extracellular matrix molecules, compose the adipose stromal vascular fraction (SVF)10. Many protocols have been reported to generate mature adipocytes from preadipocytes11,12,13,14,15 (Supplementary Table 1); nevertheless, they display extreme variations in tissue processing and the composition of the differentiation culture media. The protocol described herein allows the efficient and reproducible differentiation of brown adipocytes that (1) express the key adipogenic transcription factors peroxisome proliferator-activated receptor gamma (PPAR&#947;) and CCAAT/enhancer-binding protein alpha (C/EBP&#945;), (2) express the mature adipocyte markers perilipin1 (PLIN1), and cluster determinant 36 (CD36), (3) accumulate abundant lipid droplets, (4) have high mitochondrial mass and a high abundance of OXPHOS complexes, (5) have thermogenic potential, as determined by high levels of UCP1, and (6) have mitochondrial morphological changes associated with the phenotype of mature brown adipocytes. This methodology is used for studying the molecular mechanisms underlying generalized lipodystrophy15,16,17.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x Tris/Glycine Buffer</td>
			<td>BioRad</td>
			<td>1610734</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde Aqueous Solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm TC-treated Easy-Grip Style Cell Culture Dish</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Isobutyl-1-methylxanthine (IBMX)</td>
			<td>Calbiochem</td>
			<td>410957</td>
			<td></td>
		</tr>
		<tr>
			<td>40% Acrylamide/Bis Solution, 37.5:1</td>
			<td>BioRad</td>
			<td>1610148</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate&#160;</td>
			<td>SPL Life Science&#160;</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well optical black w/lid cell culture sterile</td>
			<td>Thermo Scientific</td>
			<td>165305</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuRuler RGB Plus Pre-stained Protein Ladder&#160;</td>
			<td>Maestrogen</td>
			<td>02102-250</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK lysing buffer&#160;</td>
			<td>Gibco</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Persulfate&#160;</td>
			<td>BioRad</td>
			<td>1610700</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic</td>
			<td>Gibco&#160;</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse IgG, HRP-linked Antibody</td>
			<td>Cell Signaling</td>
			<td>7076</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rabbit IgG, HRP-linked Antibody&#160;&#160;</td>
			<td>Cell Signaling</td>
			<td>7074</td>
			<td></td>
		</tr>
		<tr>
			<td>Blotting-grade blocker&#160;</td>
			<td>BioRad</td>
			<td>170-6404</td>
			<td></td>
		</tr>
		<tr>
			<td>BODIPY 493/503&#160;</td>
			<td>Invitrogen</td>
			<td>D3922</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A1470</td>
			<td></td>
		</tr>
		<tr>
			<td>C/EBP&#945; antibody</td>
			<td>Cell Signaling</td>
			<td>2295</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Calbiochem&#160;</td>
			<td>208291</td>
			<td></td>
		</tr>
		<tr>
			<td>CD36 antibody</td>
			<td>Invitrogen</td>
			<td>PA1-16813</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer 100 &#181;m, nylon</td>
			<td>Falcon</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer 40 &#181;m, nylon</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type II</td>
			<td>Gibco</td>
			<td>17101-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytation 5 Cell Imaging Multimode Reader</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone&#160;</td>
			<td>Sigma</td>
			<td>D4902</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, powder</td>
			<td>Gibco</td>
			<td>12500062</td>
			<td></td>
		</tr>
		<tr>
			<td>EMBed-812 EMBEDDING KIT (Epon)</td>
			<td>Electron Microscopy Sciences</td>
			<td>14120</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Merck&#160;</td>
			<td>100983&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum&#160;</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin from cold water fish skin</td>
			<td>Sigma</td>
			<td>G7041</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Gibco&#160;</td>
			<td>15023-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde 25% Aqueous Solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>16210</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594</td>
			<td>Life Technologies</td>
			<td>A11012</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Phosphatase Inhibitor Cocktail 100X&#160;</td>
			<td>Thermo Scientific</td>
			<td>78427</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Protease Inhibitor Cocktail 100X&#160;</td>
			<td>Thermo Scientific</td>
			<td>78429</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33258</td>
			<td>Invitrogen</td>
			<td>H1398</td>
			<td></td>
		</tr>
		<tr>
			<td>Immun-Blot PVDF Membrane&#160;</td>
			<td>BioRad</td>
			<td>1620177</td>
			<td></td>
		</tr>
		<tr>
			<td>Indomethacin&#160;</td>
			<td>Sigma&#160;</td>
			<td>I7378&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin&#160;</td>
			<td>Sigma</td>
			<td>I3536</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Calbiochem&#160;</td>
			<td>529552</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Calbiochem</td>
			<td>529568</td>
			<td></td>
		</tr>
		<tr>
			<td>KHCO3</td>
			<td>Sigma</td>
			<td>60339</td>
			<td></td>
		</tr>
		<tr>
			<td>Lane Marker Reducing Sample Buffer&#160;</td>
			<td>Thermo Scientific</td>
			<td>39000</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M2643</td>
			<td></td>
		</tr>
		<tr>
			<td>MilliQ water sterile&#160;</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Mitoprofile Total OXPHOS Rodent WB antibody Cocktail</td>
			<td>Abcam&#160;</td>
			<td>MS604</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Merck&#160;</td>
			<td>1064041000</td>
			<td></td>
		</tr>
		<tr>
			<td>OmniPur&#160;10x PBS Liquid Concentrate</td>
			<td>Calbiochem</td>
			<td>6505-OP</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium Tetroxide&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>19100</td>
			<td></td>
		</tr>
		<tr>
			<td>Perilipin 1 antibody</td>
			<td>Cell Signaling</td>
			<td>9349</td>
			<td></td>
		</tr>
		<tr>
			<td>PPAR&#947; (81B8) antibody</td>
			<td>Cell Signaling</td>
			<td>2443</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA buffer lysis&#160;</td>
			<td>Thermo Scientific</td>
			<td>89901</td>
			<td></td>
		</tr>
		<tr>
			<td>Rosiglitazone</td>
			<td>Merck</td>
			<td>557366</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate&#160;</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Cacpdylate</td>
			<td>Electron Microscopy Sciences</td>
			<td>12300</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate&#160;</td>
			<td>BioRad</td>
			<td>1610301</td>
			<td></td>
		</tr>
		<tr>
			<td>T3</td>
			<td>Sigma</td>
			<td>T6397</td>
			<td></td>
		</tr>
		<tr>
			<td>Talos F200C G2</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED&#160;</td>
			<td>BioRad</td>
			<td>1610800</td>
			<td></td>
		</tr>
		<tr>
			<td>TOM20 antibody</td>
			<td>Cell Signaling</td>
			<td>42406</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Buffered Saline (TBS-10X)</td>
			<td>Cell Signaling</td>
			<td>12498</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100&#160;</td>
			<td>Sigma</td>
			<td>93443</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0,25%)</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20, Molecular Biology Grade</td>
			<td>Promega&#160;</td>
			<td>H5152</td>
			<td></td>
		</tr>
		<tr>
			<td>UCP1 antibody</td>
			<td>Cell Signaling</td>
			<td>14670</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracut R</td>
			<td>Leica&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl Acetate</td>
			<td>Electron Microscopy Sciences</td>
			<td>22400</td>
			<td></td>
		</tr>
		<tr>
			<td>Westar Sun&#160;</td>
			<td>Cyanagen</td>
			<td>XLS063</td>
			<td></td>
		</tr>
		<tr>
			<td>Westar Supernova&#160;</td>
			<td>Cyanagen</td>
			<td>XLS3</td>
			<td></td>
		</tr>
	</tbody>
</table>

differentiation and imaging of brown adipocytes from the stromal vascular fraction of interscapular adipose tissue from newborn mice

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with multiple lipid droplets, a central nucleus, a high mitochondrial content, and the expression of uncoupling protein 1 (UCP1). BAT has been proposed as a potential therapeutic target for obesity and its associated metabolic disorders due to its ability to dissipate metabolic energy as heat. To investigate BAT function and regulation, brown adipocyte culturing is indispensable. The present protocol optimizes tissue processing and cell differentiation for culturing brown adipocytes from newborn mice. Additionally, procedures for the imaging of differentiated adipocytes with both confocal immunofluorescence and transmission electron microscopy are shown. In the brown adipocytes differentiated with the techniques described herein, the major defining features of classical BAT are preserved, including high UCP1 levels, increased mitochondrial mass, and very close physical contact between the lipid droplets and mitochondria, making this method a valuable tool for BAT studies.

Adipogenesis is regulated by a network of transcription factors that are responsible for both the expression of key proteins that induce brown adipocyte formation and functioning22, including classical adipogenic regulators such as PPAR&#947; and C/EBP&#945;23,24,25, as well as markers of mature adipocytes26,27. Through testing the different conce...

Watch this Scientific Journal Video about Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice at JoVE.com

Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice

Preadipocytes are isolated from the stromal vascular fraction of interscapular brown adipose tissue from newborn mice and differentiated into cells that accumulate lipid droplets, express molecular markers, and show the mitochondrial morphology of mature brown adipocytes. These cells are further analyzed by immunofluorescence and transmission electron microscopy.

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with ...

differentiation-imaging-brown-adipocytes-from-stromal-vascular

Research

JoVE Journal

Biology

1.2K Views.  Pontificia Universidad Católica de Chile. Preadipocytes are isolated from the stromal vascular fraction of interscapular brown adipose tissue from newborn mice and differentiated into cells that accumulate lipid droplets, express molecular markers, and show the mitochondrial morphology of mature brown adipocytes. These cells are further analyzed by immunofluorescence and transmission electron microscopy.

Video: Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPAR&gamma; agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies &#8211; including single-cell RNA-sequencing and single-cell proteomics &#8211; is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods are essential to provide critical insights into these thermogenic adipose depots. Here, the protocol presents a PET/MR imaging-based method to evaluate the activity of brown and beige adipocytes in mouse interscapular brown adipose tissue (iBAT) and inguinal subcutaneous white adipose tissue (iWAT). Visualization and quantification of the thermogenic adipose depots were achieved using [18F]FDG, the non-metabolizable glucose analog, as the radiotracer, when combined with the precise anatomical information provided by MR imaging. The PET/MR imaging was conducted 7 days after cold acclimation and quantitation of [18F]FDG signal in different adipose depots was conducted to assess the relative mobilization of thermogenic adipose tissues. Removal of iBAT substantially increased cold-evoked [18F]FDG uptake in iWAT of the mice.

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Functional imaging and quantitation of thermogenic adipose depots in mice using a micro-PET/MR imaging-based approach.

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods ...

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...

Adipose Tissue-Derived Stem Cell Isolation and Characterization

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

Author Spotlight: Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...

Semi-Automated Isolation of Murine White Adipose Tissue

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  

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 ...

Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice

Summary

Explore More Videos

Chapters in this video

Functional Imaging of Brown Fat in Mice with ¹⁸F-FDG micro-PET/CT