Name: differentiation and imaging of brown adipocytes from the stromal vascular fraction of interscapular adipose tissue from newborn mice
Uploaded: 2023-02-03T13:10:00
Description: 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

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 ...

<ol>
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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. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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. 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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 ...

This protocol allows the efficient and reproducible differentiation of brown adipocytes that express key adipogenic transcription factors, mature adipocyte markers, and have thermogenic potential and morphological phenotype of mature brown adipocytes. This is a simple and replicable two-phase differentiation procedure to obtain cells with characteristics of mature brown adipocytes from interscapular brown adipose tissue of newborn mice. This protocol allows studying the activation mechanisms of brown adipose tissue as a target for treating obesity.It has been a model for determining the mechanisms involved in the pathophysiology of lipodystrophy. Performing this technique for the first time is expected to be challenging. It is essential to have a detailed protocol and emphasize critical points to have successful results.To begin, place the euthanized mouse in a prone position and make a one centimeter long skin incision at the mid-level of the animal's back using sharp scissors. Remove the skin carefully, surgically detach the iBAT from the carcass using small scissors. Harvest both iBAT lobes using curve tip pliers to remove the lobes independently.Place the two lobes in a plastic tube with 1.5 milliliters of sterile PBS at room temperature. Incubate the iBAT tissues in collagenase type two digestion buffer for 45 minutes at 37 degrees Celsius with continuous gentle shaking at 800 RPM. Mechanically disrupt the tissue suspension in collagenase type two digestion buffer by pipetting up and down using a P1000 micropipette and filter tips.Use a new tip for each sample. Pass the desegregated tissues through individual 100 micrometer cell strainers into new 1.5 milliliter tubes. Add 500 microliters of ACK buffer to the filtered tissue samples.Invert the tubes four to five times and incubate at room temperature for four minutes. Then pass the suspensions through a 40 micrometer cell strainer into new 1.5 milliliter tubes using a P1000 micropipette and filtered tips. Centrifusion resuspend the pellet as described in the text manuscript.Then seed each sample into six wells of a 24 well culture plate by adding one milliliter of the suspension to each well. The undifferentiated preadipocytes presented very low or undetectable levels of ppar-gamma, C/ebp-alpha, perilipin 1 and CD36. In contrast, these markers were significantly higher on day seven of differentiation.The accumulation of multiple lipid droplets in the brown preadipocytes was consistent with the previously published results. A significant increase was observed in the mitochondrial mass marker TOM 20 and the oxidative phosphorylation complex protein levels at day seven of differentiation. UCP1, a bonafide marker of this cell type was not detected in undifferentiated preadipocytes while it was substantially expressed in mature brown adipocytes at day seven of differentiation.The mitochondria evolved from an elongated tubular shape at day zero toward a rounded or bean-like shape at day seven. The mitochondrial inner structure was also modified by adipogenesis, resulting in higher density of the parallel packed crystae. Importantly, the mitochondrial were intimately associated with lipid droplets in the differentiated brown adipocytes, so no discernible distance between the outer membrane of the mitochondria and the surfaces of the lipid droplets could be detected, even with high resolution transmission electron microscopy.The surgical removal of interscapular adipose tissue is critical. An inefficient quantity of tissue limits the availability of cells that will be differentiated. This imaging technique helps characterize the morphology of differentiated brown adipocytes in vitro.It's possible to perform assays for mitochondrial function, beta androgenic activation, and autophagic inhibition.

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

Research

JoVE Journal

Biology

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 ...

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 ...

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 ...

Culturing Preadipocytes Isolated from Murine Perivascular Adipose Tissue

Isolation, Culture, and Adipogenic Induction of Stromal Vascular Fraction-derived Preadipocytes from Mouse Periaortic Adipose Tissue

Perivascular adipose tissue (PVAT) is an adipose tissue depot that surrounds blood vessels and exhibits the phenotypes of white, beige, and brown adipocytes. Recent discoveries have shed light on the central role of PVAT in regulating vascular homeostasis and participating in the pathogenesis of cardiovascular diseases. A comprehensive understanding of PVAT properties and regulation is of great importance for the development of future therapies. Primary cultures of periaortic adipocytes are valuable for studying PVAT function and the crosstalk between periaortic adipocytes and vascular cells. This paper presents an economical and feasible protocol for the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, which can be useful for modeling adipogenesis or lipogenesis in vitro. The protocol outlines tissue processing and cell differentiation for culturing periaortic adipocytes from young mice. This protocol will provide the technological cornerstone at the bench side for the investigation of PVAT function.

Author Spotlight: The Significance of Isolation, Culture, and Adipogenic Induction of SVF-Derived Preadipocytes from Mouse Perivascular Adipose Tissue 

Here, we describe the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, allowing for the study of perivascular adipose tissue function and its relationship with vascular cells.

Perivascular adipose tissue (PVAT) is an adipose tissue depot that surrounds blood vessels and exhibits the phenotypes of white, beige, and brown ...

Isolation of Stromal Vascular Fraction (SVF) from Perivascular Adipose Tissue (PVAT)

While working under a class two laminar flow hood, begin by sequentially washing the perivascular adipose tissues or PVATs collected from mice two times. First, using PBS containing 10%penicillin-streptomycin, and, next, using PBS supplemented with 1%penicillin-streptomycin. Transfer the rinsed tissues into a sterile 60-millimeter Petri dish and add 200 microliters of digestion solution to it.Using sterile scissors, mince the tissues into pieces having a dimension of one cubic millimeter. With the help of sterile scissors, cut a plastic pipette tip to broaden its end. Then transfer the minced tissue mix to a 15-milliliter centrifuge tube using the broad-ended pipette tip.Add six milliliters of the digestion solution to the tissues to initiate digestion. After tying the tube horizontally on a rack, incubate it at 37 degrees Celsius in an orbital shaker for 30 to 45 minutes. Every 5 to 10 minutes, remove the tube from the shaker in the incubator and manually shake it up and down vigorously.Pass the digested tissue mix through a 70-micron cell strainer into a 50-milliliter centrifuge tube. Rinse the strainer with an equal volume of culture medium to maximize cell yield and quench digestion. Transfer the filtrate to a new 15-milliliter centrifuge tube.Centrifuge the tube to isolate the stromal vascular fraction or SVF. Invert the tube to discard the supernatant and re-suspend the pellet in five milliliters of PBS. Centrifuge the tube again at 1, 800 G for five minutes before discarding the supernatant.Re-suspend the pellet in an appropriate volume of culture medium. Seed the cells into a collagen-coated 12-well plate and incubate overnight at 37 degrees Celsius in a humid atmosphere with 5%carbon dioxide. On the next day, remove cell debris and red blood cells by aspirating the culture medium and washing the cells with antibiotic-supplemented PBS pre-warmed to 37 degrees Celsius.Finally, add one milliliter of fresh culture medium to each well, and continue culturing the cells until they reach the desired stage of confluency.

Dissection and Removal of the Temporal Bone to Collect Auditory Epithelia from Newborn Mice

To begin, securely hold the head of the euthanized newborn mice in place. Using micro operating scissors, open the scalp along the sagittal suture. Then, separate and immobilize the scalp bilaterally with the fingers.Use scissors to sever the cranium and flip it forward to access the skull. Employ a small periosteal elevator to extract the brain and then use the tip of the scissors to gently scrape the brain, revealing the base of the skull. Now, examine the bilateral temporal bones at the skull's base.Employ scissors to incise the base of the skull along the midline. Then, scrape off the skin and eliminate any unnecessary bone. Preserve and transfer the temporal bones to 35-millimeter sterile Petri dishes filled with fresh HBSS.Next, use a pair of 5 number pointed forceps to eliminate the bola and surrounding tissue from the petrous portion of the temporal bone. Hold the forceps with one hand to stabilize the semi-circular part of the temporal bone. Use the other hand to insert the lower tip of the forceps into the round window niche, separating the lateral bone of the cochlea from the scale of vestibule.Carefully remove the petrous portion of the temporal bone without contacting the Organ of Corti epithelium. Then, meticulously detach and extract the bony labyrinth of the cochlea, starting from the basal to the apical end. Using 5 number pointed forceps, carefully micro isolate the sensory epithelium of the Organ of Corti from the modiolus.Next, employ micro-operating forceps to hold the spiral ligament, and gently separate it from the stria vascularis. Transfer the clean auditory epithelium, using a 200 microliter pipette, to a three millimeter sterile culture dish containing HBSS. Place the isolated auditory epithelium in a 100 millimeter sterile Petri dish filled with HBSS for the subsequent preparation steps.

Morphological and Molecular Characterization of the scBAT in Mice

Dissection, Histological Processing, and Gene Expression Analysis of Murine Supraclavicular Brown Adipose Tissue

Brown adipose tissue (BAT)-mediated thermogenesis plays an important role in the regulation of metabolism, and its morphology and function can be greatly impacted by environmental stimuli in mice and humans. Currently, murine interscapular BAT (iBAT), which is located between two scapulae in the upper dorsal flank of mice, is the main BAT depot used by research laboratories to study BAT function. Recently, a few previously unknown BAT depots were identified in mice, including one analogous to human supraclavicular brown adipose tissue. Unlike iBAT, murine supraclavicular brown adipose tissue (scBAT) is situated in the intermediate layer of the neck and thus cannot be accessed as readily.
To facilitate the study of newly identified mouse scBAT, presented herein is a protocol detailing the steps to dissect intact scBAT from postnatal and adult mice. Due to scBAT&#39;s small size relative to other adipose depots, procedures have been modified and optimized specifically for processing scBAT. Among these modifications is the use of a dissecting microscope during tissue collection to increase the precision and homogenization of frozen scBAT samples to raise the efficiency of subsequent qPCR analysis. With these optimizations, the identification of, morphological appearance of, and molecular characterization of the scBAT can be determined in mice.

Author Spotlight: Optimizing Supraclavicular Brown Adipose Tissue Extraction for Genetic Analysis

Here, we provide a practical procedure for dissecting and performing histological and gene expression analyses of murine supraclavicular brown adipose tissue.

Brown adipose tissue (BAT)-mediated thermogenesis plays an important role in the regulation of metabolism, and its morphology and function can be greatly ...