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

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

Semi-Automated Isolation of Murine White Adipose Tissue

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

Here, we described a protocol for isolation of the SVF from murine adipose tissue to obtain preadipocytes and perform adipocyte differentiation in vitro. The use of a tissue dissociator for collagenase digestion decreased experimental variation, decreased the risk of contamination, and increased reproducibility. While this procedure is a critical step within the presented protocol, the process is highly automated and optimization is not needed. However, depending on the mouse age and adipose tissue depot, optimization of mincing, for example size pieces or cutting time, might be required.
The SVF is known as a heterogenous population consisting of preadipocytes, immune cells such as macrophages, endothelial cells, and other cells. Because preadipocytes adhere to culture dishes and are tolerant to washing and medium changes, they are enriched in the cell population during the passages. However, it is reasonable to assume that the &#34;preadipocytes&#34; obtained by this protocol are still heterogeneous. Fluorescent-activated cell sorting (FACS) using antibodies against previously reported surface markers of preadipocytes such as PDGFR&#945;32 would be required to obtain a purer preadipocyte population.
In summary, we described here a protocol for SVF isolation using a tissue dissociator for collagenase digestion. This protocol offers easier isolation of the SVF compared with the conventional protocol using a bacterial shaker with a tube rack, and provides reduced experimental variation, reduced contamination, and increased reproducibility16,17,18.

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

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

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

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

This protocol yields fully differentiated, lipid-laden adipocytes 7 days after inducing adipocyte differentiation. The degree of adipocyte differentiation can be evaluated by the oil red o staining of triglycerides and lipids (Figure 1A), or mRNA expression analysis using qPCR-RT of adipocyte genes, such as the master regulator of adipogenesis Pparg and its target Fabp4 (Figure 1B). To induce beige adipocyte differentiation in vitro, a thiazolidinediones class of PPAR&#947; agonist, such as rosiglitazone, can be used. Indeed, in experiments with rosiglitazone, we observe a dose-dependent effect of concentrations up to 1 &#181;M on the expression levels of the brown fat-specific genes, such as Ucp1 and Ppargc1a. On the other hand, the effect of rosiglitazone on Fabp4 is saturated at a concentration of 0.1 &#181;M (Figure 1C).
Differentiated adipocytes obtained by this protocol can be used for various functional and mechanistic analyses, such as oxygen consumption rate (OCR) analysis16,30 (Figure 2A) and chromatin immunoprecipitation31 (ChIP; Figure 2B). Immortalized preadipocytes can be stored in a liquid nitrogen cell storage system without loss of viability and can be thawed at any time for use in experiments.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65265/65265fig01.jpg" /> 
Figure 1: Differentiation of preadipocytes into lipid-laden adipocytes. (A) SVFs from inguinal white adipose tissue (iWAT) of wild-type mice were stained with oil red o at day 0 or 7 of adipocyte differentiation.(B) mRNA expression levels of Pparg and Fabp4 at day 0 and day 7 of adipocyte differentiation (mean &#177; standard error of the mean [S.E.M.]; N = 3). (C) mRNA expression of the general adipocyte marker Fabp4 and&#160;the brown fat-specific genes Ucp1 and Ppargc1a in SVF-derived adipocytes treated with 0.1, 0.2, 0.5, and 1.0 &#181;M rosiglitazone, along with the differentiation and maintenance medium. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65265/65265fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65265/65265fig02.jpg" /> 
Figure 2: Examples of functional and mechanistic analyses with differentiated adipocytes. (A) OCR analysis of iWAT SVF-derived adipocytes (N = 10). (B) ChIP-qPCR for H3K27Ac in Nfiaflox/flox iWAT SVF-derived adipocytes at day 0 and 7 of differentiation. The Ins-0.4 kb and Fabp4-13 kb site are shown as background sites (mean &#177; S.E.M.; N = 2). For both experiments, 1.0 &#181;M rosiglitazone was added along with the differentiation and maintenance medium to induce beige adipocyte differentiation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65265/65265fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Composition of differentiation and maintenance medium. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65265/Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: The list of primers used in this study. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65265/Table 2.xlsx" target="_blank">Please click here to download this Table.</a>

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

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.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Biology

2.0K Views.  The University of Tokyo. This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

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

Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of dissociating whole organs requires specific parameters in order to obtain a high number of viable cells in a reproducible manner. The gentleMACS Dissociator optimizes this task with a simple, practical protocol. The instrument contains pre-programmed settings that are optimized for the efficient but gentle dissociation of a variety of tissue types, including mouse lungs. In this publication the use of the gentleMACS Dissociator on lung tissue derived from mice is demonstrated.

Dissociating cells from specific tissue types requires specific parameters for tissue agitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on lung tissue is explained.

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of ...

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 ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Manipulating the Murine Lacrimal Gland

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for vision. In the human a single LG resides in the orbit above the lateral end of each eye delivering tears to the ocular surface through 3 - 5 ducts. The mouse has three pairs of major ocular glands, the most studied of which is the exorbital lacrimal gland (LG) located anterior and ventral to the ear. Similar to other glandular organs, the LG develops through the process of epithelial branching morphogenesis in which a single epithelial bud within a condensed mesenchyme undergoes multiple rounds of bud and duct formation to form an intricate interconnected network of secretory acini and ducts. This elaborate process has been well documented in many other epithelial organs such as the pancreas and salivary gland. However, the LG has been much less explored and the mechanisms controlling morphogenesis are poorly understood. We suspect that this under-representation as a model system is a consequence of the difficulties associated with finding, dissecting and culturing the LG. Thus, here we describe dissection techniques for harvesting embryonic and post-natal LG and methods for ex vivo culture of the tissue.

The lacrimal gland (LG) is a branching organ that produces the aqueous components of tears necessary for maintaining vision and ocular health. Here we describe murine LG dissection and ex vivo culture techniques to decipher signaling pathways involved in LG development.

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

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 ...

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

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 ...

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important players in the maintenance of organismal homeostasis and the progression of a wide spectrum of diseases. While the current research focuses on the characterization of endogenous exosomes with the endosomal origin, microvesicles blebbing from the plasma membrane have gained increasing attention in health and sickness, which are featured by an abundance of surface molecules recapitulating the membrane signature of parent cells. Here, a reproducible procedure is presented based on differential centrifugation for extracting and characterizing EVs from the plasma and solid tissues, such as the bone. The protocol further describes subsequent profiling of surface antigens and protein cargos of EVs, which are thus traceable for their derivations and identified with components related to potential function. This method will be useful for correlative, functional, and mechanistic analysis of EVs in biological, physiological, and pathological studies.

The present protocol describes a method to extract extracellular vesicles from the peripheral blood and solid tissues with subsequent profiling of surface antigens and protein cargos.

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important ...