作者要感谢Takahito Wada和Saiko Yoshida（东京大学，东京，日本）的实验帮助。这项工作由以下对Y.H.的赠款资助：东京大学优秀青年研究员计划的研究资助;日本科学促进会（JSPS）KAKENHI早期职业科学家补助金，资助号19K17976;日本应用酶学基金会未来糖尿病研究领跑者（FFDR）资助，资助号17F005;药理学研究基金会的资助;Mochida纪念医学和药物研究基金会的资助;默沙东生命科学基金会的资助;大和证券健康财团的赠款;东京生化研究财团资助;武田科学基金会生命科学研究基金;以及SENSSHIN医学研究基金会的资助。

本协议中描述的所有动物实验均由东京大学机构动物护理和使用委员会（IACUC）批准，并根据东京大学的机构指南进行。
1. 酶溶液和培养基的制备
<ol><li>将来自7-8周龄小鼠的腹股沟白色脂肪组织（右侧和左侧，约150mg）和2.5mL酶溶液放入解离器的管C中。</li>
	<li>用 3 mL 不含胎牛血清 （FBS） 或抗生素的 Dulbecco 改良鹰培养基 （DMEM）/F12 复溶酶 D，用不含胎牛血清 （FBS） 或抗生素的 2.7 mL DMEM/F12 复溶酶 R，用不含 FBS 或抗生素的酶 A 复溶酶 A，用 1 mL 缓冲液 A 复溶酶 A。</li>
	<li>通过将 100 μL 酶 D、50 μL 酶 R、12.5 μL 酶 A 和 2.35 mL 不含 FBS 或抗生素的 DMEM/F12 加入试管 C 中制备 2.5 mL 酶溶液。</li>
</ol>2.脂肪组织的分离
<ol><li>通过颈椎脱位对7-8周龄的雄性C57BL / 6J小鼠（约20g）实施安乐死。</li>
	<li>在干净的工作台上，为了分离腹股沟白色脂肪组织，用钝/锋利的剪刀在腹部和下肢切开皮肤。使用锋利/锋利的剪刀从大腿内侧分离脂肪组织。腹股沟脂肪组织的一侧~75毫克。</li>
	<li>将分离出的脂肪组织放入冰上装有酶溶液的试管C中，并将管子放在干净的工作台内以保持无菌。</li>
</ol>3.脂肪组织的切碎和消化
<ol><li>在洁净工作台上，向管C中加入2.5 mL酶溶液。</li>
	<li>用锋利/锋利的剪刀切50次，将脂肪组织切成小块。将脂肪组织切成~2毫米2 块。</li>
	<li>紧紧关闭管C的盖子，将管子倒置，并将其连接到组织解离器的套筒上，盖上盖子。然后，将样品在37°C下消化~40分钟。 注意：本研究使用带有加热器的gentleMACS Octo解离器（Miltenyi Biotec 130-096-427），并遵循预装程序“37C_mr_ATDK_1”。</li>
</ol>4. 细胞悬液的过滤
<ol><li>将含有10%FBS和1%青霉素-链霉素的DMEM/F12预热至37°C。</li>
	<li>从组织解离器中分离管 C，并通过加入含有 10% FBS 和 1% 青霉素链霉素的 5 mL DMEM/F12 并轻轻移液四次来停止消化。</li>
	<li>在20°C下以700× g 离心10分钟。</li>
	<li>小心吸出上清液而不干扰细胞沉淀。</li>
	<li>通过加入含有 10% FBS 和 1% 青霉素链霉素的 10 mL DMEM/F12 并轻轻移液 5 次来重悬沉淀。</li>
	<li>通过放置在新的 50 mL 管上的 70 μm 直径细胞过滤器过滤细胞悬液。</li>
	<li>以 250 x g 离心 5 分钟，并将沉淀重悬于 10 mL 磷酸盐缓冲盐水 （PBS） 中。</li>
</ol>5. 电池电镀
<ol><li>以500× g 离心5分钟。</li>
	<li>除去上清液，通过加入含有 10% FBS 和 1% 青霉素-链霉素的 10 mL DMEM/F12 并移液十次来重悬沉淀。</li>
	<li>将细胞悬液铺在10cm胶原蛋白包被的培养皿上，并将培养皿置于细胞培养箱（37°C，5%CO 2）中1-2小时。 注意：胶原蛋白涂层的菜肴不是绝对必需的。然而，根据经验，胶原蛋白包衣的培养皿确实有助于前脂肪细胞的粘附，从而提高细胞的存活率。</li>
	<li>吸出培养基，每次洗涤用 3 mL PBS 洗涤细胞两次。</li>
	<li>加入含有10%FBS和1%青霉素链霉素的10mL DMEM / F12，并将培养皿放回细胞培养箱（37°C，5%CO2）。</li>
</ol>6. 前脂肪细胞的传代
<ol><li>第二天，吸出培养基（细胞将贴壁，因此可以吸出培养基），每次洗涤用 3 mL PBS 洗涤细胞两次，并加入 10 mL 含有 10% FBS 和 1% 青霉素-链霉素的 DMEM/F12。</li>
	<li>当细胞达到80%汇合时（通常在SVF分离后4天），将细胞分成四个10厘米胶原蛋白包被的培养皿。<ol><li>具体来说，吸出培养基，用PBS（室温[RT]）洗涤细胞，加入1mL预热的0.05%胰蛋白酶，并在细胞培养箱中孵育5分钟。</li>
		<li>加入含有 10% FBS 和 1% 青霉素-链霉素的 10 mL DMEM/F12 以淬灭胰蛋白酶活性。移液后，以440 x g 离心5分钟。</li>
		<li>吸出上清液，通过加入含有 10% FBS 和 1% 青霉素链霉素的 40 mL DMEM/F12 重悬细胞，并将细胞接种在四个 10 cm 胶原蛋白包衣的培养皿中。</li>
	</ol></li>
	<li>当细胞达到80%汇合时再次传代细胞。</li>
</ol>7.制备表达SV40大T抗原的逆转录病毒，用于前脂肪细胞永生化（可选）
<ol><li>在永生化前至少 3 天，将 Platinum-E （Plat-E） 细胞29 以 3.0 x 105 个细胞 /mL 的密度包装在 2 mL 的 DMEM 中，加入不含抗生素的 10% FBS。使用血细胞计数器计数细胞。</li>
	<li>第二天，根据制造商的说明，使用Lipofectamine 2000转染4μgpBABE-neo largeTcDNA（添加基因质粒#1780）。</li>
	<li>24小时后，吸出培养基并加入2mL DMEM和10%FBS和1%青霉素 - 链霉素。</li>
	<li>第二天，收获逆转录病毒上清液。逆转录病毒可立即用于永生化或储存在-80°C。</li>
</ol>8.用SV40大T抗原使前脂肪细胞永生化（可选）
<ol><li>SVF分离后传代并扩增前脂肪细胞两次。</li>
	<li>将细胞以 0.5-1.0 x 10 的密度将细胞接种在 6 孔板中4 个细胞/mL 的 2 mL DMEM/F12 中，含有 10% FBS 和 1% 青霉素-链霉素。使用血细胞计数器计数细胞。</li>
	<li>第二天，在 6 孔板的每孔中制备 250 μL 表达 SV40 大 T 抗原的新鲜或解冻逆转录病毒。以440× g 离心5分钟，并将上清液放入新管中。</li>
	<li>每 250 μL 逆转录病毒上清液中加入 750 μL 含有 10% FBS 和 1% 青霉素-链霉素和 1 μL 聚溴乙烯的 DMEM/F12，制成工作病毒。</li>
	<li>吸出培养基，并向每个含有前脂肪细胞的孔中加入 1，000 μL 工作逆转录病毒。</li>
	<li>4 小时后加入 1 mL DMEM/F12 和 10% FBS 和 1% 青霉素-链霉素。</li>
	<li>第二天，将感染的前脂肪细胞分离到10厘米的培养皿中，并用含有10%FBS，1%青霉素 - 链霉素和500μg/ mL新霉素的DMEM / F12选择感染细胞。为了监测抗生素的选择，用新霉素准备一个未感染细胞的培养皿。</li>
	<li>继续用新霉素传代感染的细胞，直到监测培养皿中所有未感染的细胞都死亡。</li>
</ol>9.脂肪细胞分化
<ol><li>将细胞铺板。对于 12 孔板，将细胞以 4.0 x 104 个细胞 /mL 的密度接种在含有 10% FBS 和 1% 青霉素-链霉素的 1 mL DMEM/F12 中。</li>
	<li>当前脂肪细胞汇合时（铺板后48-72小时），吸出培养基并用分化培养基代替（组成见 表1 ）。</li>
	<li>在分化的第2天（即诱导分化后48小时），用维持培养基替换培养基（成分见 表1 ）。之后，每 2 天更换一次维护介质。在分化的第 7 天实现末端分化。</li>
	<li>油红o染色<ol><li>对于油红o染色，吸出培养基并用每孔1mL PBS冲洗细胞。通过每孔加入 1 mL 4% 甲醛来固定细胞，并在室温下孵育细胞 15 分钟。</li>
		<li>在孵育期间，使用ddH 2O将0.5%油红O储备溶液（在异丙醇中）稀释至0.3%，并使用滤纸过滤工作溶液。</li>
		<li>孵育15分钟后，除去甲醛，用60%异丙醇冲洗细胞，加入1mL过滤油红o工作溶液，在室温下孵育1小时。</li>
		<li>孵育后，用流水洗涤细胞。然后，在倒置显微镜下观察富含脂质的脂肪细胞（放大倍率：20倍物镜和10倍目镜）。</li>
	</ol></li>
	<li>mRNA表达分析 注意：有关本研究中使用的引物列表，请参阅 表2 。<ol><li>吸出培养基并向孔中加入 1 mL/孔的曲唑。</li>
		<li>在室温下孵育15分钟，通过移液分离细胞，并将样品收集在1.5mL管中。样品可以储存在-80°C直到RNA提取。</li>
		<li>将冷冻样品解冻至室温，向样品中加入 200 μL 氯仿，剧烈摇动 15 秒。</li>
		<li>将样品在室温下孵育3分钟，然后在4°C下以20，000× g 旋转15分钟。</li>
		<li>小心地去除水相（顶部，无色）并转移到新管中。切勿转移蛋白质相（中间，白色）或苯酚/氯仿相（底部，粉红色）。</li>
		<li>慢慢加入等体积的70%EtOH并轻轻混合。不要涡旋。</li>
		<li>将样品（最多 700 μL）加载到位于收集管中的 RNA 离心柱中。确保包括可能已经形成的任何沉淀物。</li>
		<li>在4°C下以9，000× g 旋转30秒，然后丢弃流通物。</li>
		<li>加入 700 μL 缓冲液 RW1，在 4 °C 下以 9，000 x g 离心 30 秒，然后丢弃流通物。</li>
		<li>将色谱柱转移到新的收集管中，并加入500 μL缓冲液RPE。</li>
		<li>在4°C下以9，000× g 旋转30秒，然后丢弃流出物。</li>
		<li>加入 500 μL 缓冲液 RPE，在 4 °C 下以 9，000 x g 离心 2 分钟，然后弃去流通物。</li>
		<li>将色谱柱转移到新的收集管中，并在4°C下以9，000× g 旋转（无缓冲液的柱）1-2分钟。</li>
		<li>将色谱柱转移到新的 1.5 mL 收集管中，并将 30 μL 无 RNase 的水直接添加到柱膜上。</li>
		<li>将样品在室温下孵育1-2分钟。然后，在4°C下以9，000× g 旋转1分钟以洗脱RNA。</li>
		<li>使用透光谱仪检查RNA浓度。 注意：建议在此处对齐浓度。</li>
		<li>使用 ~200 ng 的 RNA 模板进行逆转录。使用商用定量实时聚合酶链反应 （qPCR-RT） 试剂盒并遵循制造商的方案。反应后，将cDNA稀释20倍至200μL。</li>
		<li>向 384 孔板的每个孔中加入 2.0 μL cDNA、3.0 μL Power Sybr Green 预混液、0.018 μL 100 μM qPCR 正向引物、0.018 μL 100 μM qPCR 反向引物和 0.96 μL ddH 2 O。</li>
		<li>运行qPCR（保持阶段：50°C2分钟和95°C2分钟;PCR阶段：40个循环，95°C持续15秒，60°C循环1分钟;熔融曲线阶段：95°C15秒，60°C1分钟，95°C15秒）。使用标准曲线法进行定量。</li>
	</ol></li>
</ol>

小鼠白色脂肪组织的半自动分离

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+subtype-selective+recruitment+of+TLE3+or+Prdm16+by+PPAR&#947;+specifies+lipid+storage+versus+thermogenic+gene+programs.">Adipose subtype-selective recruitment of TLE3 or Prdm16 by PPAR&#947; specifies lipid storage versus thermogenic gene programs.</a> Cell Metabolism. 17 (3), 423-435 (2013).</li><li>Pearson, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+TLE3+promotes+the+mitochondrial+program+in+beige+adipocytes+and+improves+glucose+metabolism.">Loss of TLE3 promotes the mitochondrial program in beige adipocytes and improves glucose metabolism.</a> Genes and Development. 33 (13-14), 747-762 (2019).</li><li>Shao, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zfp423+maintains+white+adipocyte+identity+through+suppression+of+the+beige+cell+thermogenic+gene+program.">Zfp423 maintains white adipocyte identity through suppression of the beige cell thermogenic gene program.</a> Cell Metabolism. 23 (6), 1167-1184 (2016).</li><li>Green, H., Kehinde, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sublines+of+mouse+3T3+cells+that+accumulate+lipid.">Sublines of mouse 3T3 cells that accumulate lipid.</a> Cell. 1 (3), 113-116 (1974).</li><li>Green, H., Kehinde, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+established+preadipose+cell+line+and+its+differentiation+in+culture+II.+Factors+affecting+the+adipose+conversion.">An established preadipose cell line and its differentiation in culture II. Factors affecting the adipose conversion.</a> Cell. 5 (1), 19-27 (1975).</li><li>Green, H., Kehinde, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+heritable+changes+leading+to+increased+adipose+conversion+in+3T3+cells.">Spontaneous heritable changes leading to increased adipose conversion in 3T3 cells.</a> Cell. 7 (1), 105-113 (1976).</li><li>Aune, U. L., Ruiz, L., Kajimura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+differentiation+of+stromal+vascular+cells+to+beige/brite+cells.">Isolation and differentiation of stromal vascular cells to beige/brite cells.</a> Journal of Visualized Experiments. (73), e50191 (2013).</li><li>Galmozzi, A., Kok, B. P., Saez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+differentiation+of+primary+white+and+brown+preadipocytes+from+newborn+mice.">Isolation and differentiation of primary white and brown preadipocytes from newborn mice.</a> Journal of Visualized Experiments. (167), e62005 (2021).</li><li>Priya, N., Sarcar, S., Majumdar, A. S., SundarRaj, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explant+culture:+a+simple,+reproducible,+efficient+and+economic+technique+for+isolation+of+mesenchymal+stromal+cells+from+human+adipose+tissue+and+lipoaspirate.">Explant culture: a simple, reproducible, efficient and economic technique for isolation of mesenchymal stromal cells from human adipose tissue and lipoaspirate.</a> Journal of Tissue Engineering and Regenerative Medicine. 8 (9), 706-716 (2014).</li><li>Lockhart, R. A., Aronowitz, J. A., Dos-Anjos Vilaboa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+freshly+isolated+human+adipose+stromal+cells+for+clinical+applications.">Use of freshly isolated human adipose stromal cells for clinical applications.</a> Aesthetic Surgery Journal. 37, S4-S8 (2017).</li><li>Morita, S., Kojima, T., Kitamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plat-E:+an+efficient+and+stable+system+for+transient+packaging+of+retroviruses.">Plat-E: an efficient and stable system for transient packaging of retroviruses.</a> Gene Therapy. 7 (12), 1063-1066 (2000).</li><li>Li, Y., Fromme, T., Klingenspor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meaningful+respirometric+measurements+of+UCP1-mediated+thermogenesis.">Meaningful respirometric measurements of UCP1-mediated thermogenesis.</a> Biochimie. 134, 56-61 (2017).</li><li>Hiraike, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+immunoprecipitation+with+mouse+adipocytes+using+hypotonic+buffer+to+enrich+nuclear+fraction+before+fixation.">Chromatin immunoprecipitation with mouse adipocytes using hypotonic buffer to enrich nuclear fraction before fixation.</a> STAR Protocols. 4 (1), 102093 (2023).</li><li>Rodeheffer, M. S., Birsoy, K., Friedman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+white+adipocyte+progenitor+cells+in+vivo.">Identification of white adipocyte progenitor cells in vivo.</a> Cell. 135 (2), 240-249 (2008).</li></ol>

没有一个作者与这项工作有竞争性的经济利益。

在这里，我们描述了一种从小鼠脂肪组织中分离SVF以获得前脂肪细胞并在 体外进行脂肪细胞分化的方案。使用组织解离剂进行胶原酶消化减少了实验变异，降低了污染风险，并提高了可重复性。虽然此过程是所提出的协议中的关键步骤，但该过程是高度自动化的，不需要优化。但是，根据小鼠年龄和脂肪组织库，可能需要优化切碎，例如大小碎片或切割时间。
SVF 被称...

由于全球肥胖和2型糖尿病的患病率日益增加，脂肪组织生物学一直受到越来越多的关注1。脂肪细胞以脂滴的形式储存多余的能量，这些脂滴在饥饿时释放。此外，脂肪组织通过充当内分泌器官并与其他组织交流来维持全身能量稳态2，3。有趣的是，过多的脂肪组织（肥胖）和脂肪损失（脂肪营养不良）都与胰岛素抵抗和糖尿病有关1。脂肪细胞分为三种类型：白色、棕色和米色1.白色脂肪细胞主要以脂质的形式储存多余的能量，而棕色和米色的脂肪细胞通过线粒体解偶联蛋白-1（Ucp1）以热量的形式耗散能量1，4。值得注意的是，米色脂肪细胞（也称为“诱导”棕色脂肪细胞）响应寒冷或交感神经刺激出现在白色脂肪组织中，并表现出与“经典”棕色脂肪细胞重叠但不同的基因表达模式5。最近，棕色和米色脂肪细胞有望成为抗肥胖和抗糖尿病治疗的潜在靶标，旨在“增强能量耗散”而不是“抑制能量摄入”4。支持性地，FTO肥胖变体rs1421085在人类中的风险等位基因，在常见变异6，7中表现出与较高体重指数（BMI）的最强关联，并表现出各种基因 - 环境相互作用8，9，据报道对米色脂肪细胞分化和功能产生负调节10.过氧化物酶体增殖物激活受体γ（PPARγ）被称为脂肪生成的主要转录调节因子，对于脂肪细胞分化是必要且充分的11。转录调节因子，例如含有 16 （PRDM16）、早期 B 细胞因子 2 （EBF2） 和核因子 I-A （NFIA） 的 PRD1-BF1-RIZ1 同源结构域，对于棕色和米色脂肪细胞分化和功能至关重要 12，13，14，15，16，17，18.另一方面，白色脂肪细胞基因编程需要转录调节因子，例如转导蛋白样增强蛋白3（TLE3）和锌指蛋白423（ZFP423）19，20，21。
体外模型系统能够进行分子研究，旨在提高对脂肪细胞功能和功能障碍机制的理解。尽管存在公开可用且永生化的前脂肪细胞系，例如3T3-L1和3T3-F442A222222，但原代前脂肪细胞的培养和分化为脂肪细胞将是更适合研究体内脂肪生成的模型。从小鼠脂肪组织中分离基质血管部分（SVF）是获得原代前脂肪细胞25，26的公知方法。然而，脂肪组织的胶原酶消化通常使用带有试管架的细菌摇床进行，可导致实验变化，并且容易受到污染27，28。在这里，我们描述了一种替代方案，该方案使用温和的磁激活细胞分选（MACS）组织解离剂进行胶原酶消化，以实现更容易的SVF分离。

<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

白色、棕色和米色脂肪细胞分化的 体外 研究能够研究脂肪细胞的细胞自主功能及其机制。永生化的白色前脂肪细胞系是公开的，并被广泛使用。然而，使用公开的白色脂肪细胞系很难充分概括白色脂肪细胞响应外部线索在白色脂肪组织中出现米色脂肪细胞。通常从小鼠脂肪组织中分离基质血管部分（SVF）以获得原代前脂肪细胞并进行脂肪细胞分化。然而，用手切碎和胶原酶消化脂肪组织会导致实验变化，并且容易受到污染。在这里，我们提出了一种改进的半自动方案，该方案利用组织解离剂进行胶原酶消化，以实现更容易的SVF分离，目的是减少实验变异，减少污染并提高可重复性。获得的前脂肪细胞和分化的脂肪细胞可用于功能和机理分析。

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.

作者聚焦：使用组织解离剂从小鼠白色脂肪组织中半自动分离基质血管组分  

使用组织解离器从小鼠白色脂肪组织中半自动分离基质血管部分

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

该方案在诱导脂肪细胞分化后 7 天产生完全分化的、富含脂质的脂肪细胞。脂肪细胞分化的程度可以通过甘油三酯和脂质的油红o染色（图1A）或使用脂肪细胞基因的qPCR-RT进行mRNA表达分析来评估，例如脂肪生成Pparg的主调节因子及其靶标Fabp4（图1B）。为了在体外诱导米色脂肪细胞分化，可以使用一类噻唑烷二酮类PPARγ激动剂，例如?...

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

该协议描述了从小鼠脂肪组织中半自动分离基质血管部分（SVF）以获得前脂肪细胞并在 体外实现脂肪细胞分化。使用组织解离剂进行胶原酶消化可减少实验变异并提高可重复性。

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

我们对脂肪细胞的棕色和米色亚型感兴趣，它们以热量的形式耗散能量，而白色脂肪细胞通常储存能量 我们的目标是开发一种基于促进能量消耗而不是抑制能量摄入的肥胖和糖尿病的新疗法。我们已经通过全基因组开放染色质分析确定了转录因子NFI作为棕色脂肪细胞分化的关键调节因子。NFI和脂肪生成的主要转录调节因子PPAR-γ共同定位为棕色脂肪特异性增强子。在从小鼠脂肪组织中分离基质血管部分的过程中，手动切碎和消化脂肪组织的胶原酶会导致实验变异性和污染。我们的方法使用组织解离剂进行胶原酶消化，这有助于SVF分离，减少实验变异性和污染，并提高可重复性。使用个体分化的脂肪细胞进行实验对于我们领域的研究是必不可少的。我们的方案将为随后的脂肪细胞分化提供简单且可重复的SVF分离，并将加速我们社区的研究。目前，我们正在研究NFI对全身代谢（如体重和葡萄糖）的作用，使用功能增益和丧失小鼠模型。我们还将在不久的将来探索NFI对每个下游途径的其他药理学激活。

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

Research

JoVE Journal

Biology

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

1.5K 次观看.  The University of Tokyo. 我们对脂肪细胞的棕色和米色亚型感兴趣，它们以热量的形式耗散能量，而白色脂肪细胞通常储存能量 我们的目标是开发一种基于促进能量消耗而不是抑制能量摄入的肥胖和糖尿病的新疗法。我们已经通过全基因组开放染色质分析确定了转录因子NFI作为棕色脂肪细胞分化的关键调节因子。NFI和脂肪生成的主要转录调节因子PPAR-γ共同定位为棕色脂肪特异性增强子。在从?...

视频: 作者聚焦：使用组织解离剂从小鼠白色脂肪组织中半自动分离基质血管组分  

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 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.

科研

生物学

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

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