著者は、優れた技術支援を提供してくれたLisa HansenとKirsten Vestergaard、そして原稿の批判的な読み方をしてくれたP. Scherer、N. Joffin、C. Creweに感謝しています。著者らは、ここで説明するプロトコルの開発における優れたガイダンスと支援を提供してくれたUTSW Flow Cytometry Coreに感謝します。R.K.G. は、NIH NIDDK R01 DK104789、NIDDK RC2 DK118620、および NIDDK R01 DK119163 でサポートされています。J.P.は、Innovation Fund Denmarkから博士課程前賞を受賞しています。

すべての動物のプロトコルと手順は、テキサス大学サウスウェスタンメディカルセンターの施設用動物使用およびケア委員会によって承認されています。
1. 性腺白脂肪組織からの間質血管画分(SVF)の単離
<ol><li>6〜8週齢のマウスの性腺白脂肪組織を解剖し、脂肪パッドを1x PBS溶液に入れます。</li>
	<li>最大4つの脂肪デポ(1〜2匹のマウスから2〜4のデポを推奨)を組み合わせ、200 μLの消化バッファー(1x HBSS、1.5% BSAおよび1 mg/mLコラゲナーゼD)を含む10 mLビーカーで組織をミンチにします。</li>
	<li>ミンチ組織を10 mL消化バッファーが入った50 mLの遠心チューブに移します。</li>
	<li>混合物を37°Cの水浴中で1時間、振とうしながらインキュベートします。</li>
	<li>100 μmのセルストレーナーで消化した組織をろ過し、未消化の組織を除去します。</li>
	<li>ろ過したサンプルを 2% FBS を含む PBS で 30 mL に希釈し、600 x g で 4 °C で 5 分間遠心分離します。 成熟脂肪細胞を含む上清を吸引します。</li>
	<li>すぐに優先細胞単離法に進んでください。</li>
</ol>2. FACSを用いたAPCとFIPの分離
<ol><li>チューブを穏やかに振って、ペレットを1x RBC溶解バッファー1 mLに溶解します。</li>
	<li>室温で1〜2分間インキュベートします。</li>
	<li>2% FBS/PBSを10 mL加え、40 μmのセルストレーナーを清浄な50 mLの遠心チューブに通します。</li>
	<li>600 x g で4°Cで5分間遠心分離します。</li>
	<li>培地を吸引し、ペレットを400〜800μLの2%FBS/PBS含有Fcブロック(1:200)に再懸濁します。</li>
	<li>4°Cで10分間インキュベートします。</li>
	<li>1mLあたり6細胞を≤10個含有する細胞懸濁液400μL〜800μLを1.5mLチューブに移し、抗体を添加します。抗体濃度は、材料表のセクションに記載されています。<ol><li>1)未染色細胞、2)単色コントロール、3)FMO(蛍光マイナス1)コントロール用に別々のコントロールチューブを調製します。</li>
		<li>PDGFRβ、CD45、CD31、CD9、LY6C抗体でサンプルを染色します。</li>
	</ol></li>
	<li>4°Cで15分間インキュベートし、光から保護します。</li>
	<li>600 x g で4°Cで5分間遠心分離します。</li>
	<li>培地を吸引し、細胞を400 μLの2% FBS/PBSに再懸濁します。</li>
	<li>600 x g で4°Cで5分間遠心分離します。</li>
	<li>培地を吸引し、細胞を400-800 μLの2% FBS/PBSに再懸濁します。次に、40 μmのフィルターキャップを5 mLポリスチレン丸底チューブに入れ、FACS用に入れます。</li>
	<li>ゲートについては、次の手順に従います。<ol><li>補正には、汚れていない単色コントロールを使用します。</li>
		<li>FMOコントロールを使用して実験ゲートを設定します。</li>
		<li>図 1 に示すゲーティング戦略を使用して、APC と FIPS を取得します。ゲートAPCはCD45-CD31-PDGFRβ+ LY6C-CD9-、FIPはCD45-CD31-PDGFRβ+ LY6C+細胞です。</li>
	</ol></li>
	<li>選別した細胞を500 μLの100%血清が入ったチューブに集め、細胞を氷上に保持します。</li>
	<li>細胞を600 x g で5分間、4°Cで遠心分離します。 2% FBS/PBSを添加し、再度600 x g で4°C、5分間遠心分離して細胞を洗浄します。 上清を捨て、ペレット化した細胞を使用します。</li>
	<li>ペレット化した細胞を、適切な量の性腺APC培養培地(細胞培養用)またはその後の分析のために適切な緩衝液に再懸濁します。</li>
</ol>3. 脂肪形成画分と非脂肪形成画分の免疫磁気分離
<ol><li>ステップ1.1-1.7に従って、性腺白脂肪組織からSVFを単離します。</li>
	<li>上清を吸引し、SVFペレットを1x 1x 市販のMS Bufferに10 mL再懸濁します。</li>
	<li>細胞懸濁液を40μmの細胞ストレーナーでろ過します。</li>
	<li>600 x g で4°Cで5分間遠心分離します。</li>
	<li>上清を吸引し、ペレット化した細胞を1x MS Bufferの100 μLに再懸濁します。</li>
	<li>以下で説明するように、CD31+ およびCD45+ 細胞の枯渇を行います(図2A、 ステップ1)。これは、内皮細胞と造血系統細胞を除去するために行われます。<ol><li>セルカウンターを使用して細胞をカウントし、濃度を 1 x 108 細胞/mL ≤に調整します。</li>
		<li>ビオチン標識CD31抗体とCD45抗体を細胞懸濁液にそれぞれ10個6個あたり ≤0.25μgの濃度で加え、氷上で15分間インキュベートします。</li>
		<li>1x MS Bufferを4 mL添加して細胞を洗浄します。</li>
		<li>600 x g で4°Cで5分間遠心分離します。</li>
		<li>上清を吸引し、ペレットを1x MSバッファー100 μLで再懸濁します。</li>
		<li>ストレプトアビジンナノビーズ10μLを添加し、細胞を氷上で15分間インキュベートします。</li>
		<li>1x MSバッファー4 mLを加えて細胞を洗浄します。</li>
		<li>600 x g で4°Cで5分間遠心分離し、上清を吸引します。</li>
		<li>細胞を2.5 mLの1x MSバッファーに再懸濁し、5 mLのポリプロピレンチューブに移します。</li>
		<li>チューブをマグネットラックに5分間置きます。</li>
		<li>細胞懸濁液を清潔な15 mL遠心チューブに注ぎ、非標識細胞を回収します。 注:ぶら下がっている液滴を振ったり吸い取ったりすると、不要な細胞分画による汚染につながるため、避けてください。</li>
		<li>チューブを磁石から取り外し、手順3.6.9を繰り返します。を 3.6.11.さらに2回、合計3回の洗浄。</li>
	</ol></li>
	<li>脂肪形成画分と非脂肪形成画分の分離(図2A、 ステップ2)<ol><li>ラベルのない画分(つまり、CD45-CD31-画分)で作業を続けます。</li>
		<li>非標識細胞を含む15 mLチューブを600 x g で4°Cで5分間遠心分離します。</li>
		<li>上清を吸引し、ペレットを1x MSバッファー100 μLに再懸濁します。</li>
		<li>ビオチン標識LY6C抗体とCD9抗体をそれぞれ≤ 0.25 μg/106 細胞あたり添加し、氷上で15分間インキュベートします。</li>
		<li>手順 3.6.3 に従います。を 3.6.12.をクリックして、2 回目の分離を完了します。</li>
		<li>バインドされていない分数を収集します。非標識細胞(LY6C-CD9-)は、APCを含む脂肪形成画分を表します(図2A、ステップ3)。</li>
		<li>チューブを磁石から取り出し、結合細胞を再懸濁して1x MSバッファーで溶出します。溶出液または標識画分(LY6C+ CD9+)は、FIPを含む非脂肪形成画分を表します(図2A、 ステップ3)。</li>
		<li>標識フラクションおよび非標識フラクションを600 x g で5分間遠心分離し、細胞を4°Cでペレット化します。</li>
	</ol></li>
	<li>直ちに細胞培養に進むか(ステップ6)、または優先分析のために未分化前駆体を使用します(ステップ4および/またはステップ5)。</li>
</ol>4. 脂肪形成画分および非脂肪生成画分の純度をフローサイトメトリーで評価する
<ol><li>ビーズ単離した細胞画分を、200-400 μLの2% FBS/PBS含有Fcブロック(1:200)に再懸濁します。</li>
	<li>4°Cで10分間インキュベートします。</li>
	<li>細胞懸濁液を1.5mLチューブに移し、抗体を添加します。抗体濃度は 材料表に記載されています。<ol><li>1)未染色細胞、2)単色コントロール、3)FMOコントロール用に別々のコントロールチューブを調製します。</li>
		<li>サンプルにCD45、CD31、CD9、およびLY6C抗体を添加します。</li>
	</ol></li>
	<li>4°Cで15分間インキュベートし、光から保護します。</li>
	<li>600 x g で4°Cで5分間遠心分離します。</li>
	<li>上清を吸引し、細胞を400 μLの2% FBS/PBSに再懸濁します。</li>
	<li>懸濁液を600 x g で4°Cで5分間遠心分離します。</li>
	<li>上清を吸引し、細胞を400-800μLの2%FBS/PBSに再懸濁します。40 μmのフィルターキャップを通して5 mLのポリスチレン丸底チューブにろ過し、フローサイトメトリーで分析します。</li>
	<li>純度を評価するためのフローサイトメトリー解析<ol><li>補正には、汚れていない単色コントロールを使用します。</li>
		<li>FMOコントロールを使用して実験ゲートを設定します。</li>
		<li>図2Bに示すように、生細胞と単一細胞にゲーティング戦略を使用します。</li>
		<li>CD45、CD31、LY6C、および CD9 について 、図 2B に示すようにゲーティングを実行します。</li>
		<li>フローサイトメトリーソフトウェアを使用して、単離画分中のCD45-CD31-LY6C-CD9-細胞(APC)およびCD45-CD31-LY6C+細胞(FIP)の頻度を評価します。</li>
	</ol></li>
</ol>5. FIPおよびAPCの純度を評価するための定量PCRを用いた遺伝子発現解析
<ol><li>磁気的に単離された細胞またはFACSで単離された細胞から、市販の抽出キット( 材料表を参照)を使用して、製造元の指示に従ってmRNAを抽出します。</li>
	<li>1 μg の RNA を使用して、製造元の指示に従って cDNA 逆転写酵素キット ( 材料表を参照) を使用して cDNA を合成します。</li>
	<li>APCおよびFIP選択的遺伝子の相対的なmRNAレベル(表1)を、SYBRグリーンPCRマスターミックスを用いた定量的PCRにより測定します。<ol><li>PCR用のサンプル反応を次のように設定します:5 μLの2x SYBR緑色色素、1 μLのcDNA(~50 ng/μL)、各フォワードプライマーとリバースプライマー0.5 μL(10 μM)、および3 μLの水。</li>
		<li>定量的PCRランには、以下の標準PCR条件を使用してください: ホールドステージ:50°Cで2分間、95°Cで10分間;PCRステージ(40サイクル):95°Cで15秒、60°Cで1分間。</li>
	</ol></li>
	<li>ΔΔ-Ctを計算することにより、値をハウスキーピング遺伝子(Rps18)レベルに正規化します。</li>
	<li>統計的有意性を評価するには、対応のないスチューデントのt検定を実行します。</li>
</ol>6. 細胞培養と分化
<ol><li>磁気的に単離された細胞またはFACSで、600 x g で4°Cで5分間遠心分離します。</li>
	<li>ペレットを500 μLの性腺APC培養培地に再懸濁し、48ウェル培養プレートの各画分から40K細胞/ウェルをプレート化します。</li>
	<li>メディアは1〜2日ごとに交換してください。APCは、合流点に近づくにつれて脂肪細胞への分化が始まります。同じ培地で維持されているFIPは、脂肪形成を受けることに耐性があります。</li>
</ol>

<ol>
	<li>Ouchi, N., Parker, J. L., Lugus, J. J., Walsh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipokines+in+inflammation+and+metabolic+disease.">Adipokines in inflammation and metabolic disease.</a> Nature Reviews Immunology. 11 (2), 85-97 (2011).</li><li>Rosen, E. D., Spiegelman, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+we+talk+about+when+we+talk+about+fat.">What we talk about when we talk about fat.</a> Cell. 156 (1-2), 20-44 (2014).</li><li>Funcke, J. B., Scherer, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+adiponectin+and+leptin:+adipose+tissue-derived+mediators+of+inter-organ+communication.">Beyond adiponectin and leptin: adipose tissue-derived mediators of inter-organ communication.</a> Journal of Lipid Research. 60 (10), 1648-1684 (2019).</li><li>Eto, H., 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+structure+and+cellular+components+of+aspirated+and+excised+adipose+tissue.">Characterization of structure and cellular components of aspirated and excised adipose tissue.</a> Plastic and Reconstructive Surgery. 124 (4), 1087-1097 (2009).</li><li>Hirsch, J., Batchelor, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+cellularity+in+human+obesity.">Adipose tissue cellularity in human obesity.</a> Clinics in Endocrinology and Metabolism. 5 (2), 299-311 (1976).</li><li>Ghaben, A. L., Scherer, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipogenesis+and+metabolic+health.">Adipogenesis and metabolic health.</a> Nature Reviews Molecular Cell Biology. 20 (4), 242-258 (2019).</li><li>Hepler, C., Gupta, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expanding+problem+of+adipose+depot+remodeling+and+postnatal+adipocyte+progenitor+recruitment.">The expanding problem of adipose depot remodeling and postnatal adipocyte progenitor recruitment.</a> Molecular Cell Endocrinology. 445, 95-108 (2017).</li><li>Jo, 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=Hypertrophy+and/or+Hyperplasia:+Dynamics+of+Adipose+Tissue+Growth.">Hypertrophy and/or Hyperplasia: Dynamics of Adipose Tissue Growth.</a> PLoS Computational Biology. 5 (3), 1000324 (2009).</li><li>Sun, K., Kusminski, C. M., Scherer, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+remodeling+and+obesity.">Adipose tissue remodeling and obesity.</a> Journal of Clinical Investigation. 121 (6), 2094-2101 (2011).</li><li>Kloting, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-sensitive+obesity.">Insulin-sensitive obesity.</a> American Journal of Physiology-Endocrinology and Metabolism. 299 (3), 506-515 (2010).</li><li>Vishvanath, L., Gupta, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+adipogenesis+to+healthy+adipose+tissue+expansion+in+obesity.">Contribution of adipogenesis to healthy adipose tissue expansion in obesity.</a> Journal of Clinical Investigation. 129 (10), 4022-4031 (2019).</li><li>Choe, S. S., Huh, J. Y., Hwang, I. J., Kim, J. I., Kim, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+Tissue+Remodeling:+Its+Role+in+Energy+Metabolism+and+Metabolic+Disorders.">Adipose Tissue Remodeling: Its Role in Energy Metabolism and Metabolic Disorders.</a> Frontiers in Endocrinology (Lausanne). 7, 30 (2016).</li><li>Hepler, C., Vishvanath, L., Gupta, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sorting+out+adipocyte+precursors+and+their+role+in+physiology+and+disease.">Sorting out adipocyte precursors and their role in physiology and disease.</a> Genes and Development. 31 (2), 127-140 (2017).</li><li>Burl, R. B., 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=Deconstructing+Adipogenesis+Induced+by+beta3-Adrenergic+Receptor+Activation+with+Single-Cell+Expression+Profiling.">Deconstructing Adipogenesis Induced by beta3-Adrenergic Receptor Activation with Single-Cell Expression Profiling.</a> Cell Metabolism. 28 (2), 300-309 (2018).</li><li>Hepler, 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=Identification+of+functionally+distinct+fibro-inflammatory+and+adipogenic+stromal+subpopulations+in+visceral+adipose+tissue+of+adult+mice.">Identification of functionally distinct fibro-inflammatory and adipogenic stromal subpopulations in visceral adipose tissue of adult mice.</a> Elife. 7, 39636 (2018).</li><li>Merrick, D., 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=Identification+of+a+mesenchymal+progenitor+cell+hierarchy+in+adipose+tissue.">Identification of a mesenchymal progenitor cell hierarchy in adipose tissue.</a> Science. 364 (6438), 2501 (2019).</li><li>Schwalie, P. 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=A+stromal+cell+population+that+inhibits+adipogenesis+in+mammalian+fat+depots.">A stromal cell population that inhibits adipogenesis in mammalian fat depots.</a> Nature. 559 (7712), 103-108 (2018).</li><li>Bagchi, D. P., MacDougald, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+Dissection+of+Diverse+Mouse+Adipose+Depots.">Identification and Dissection of Diverse Mouse Adipose Depots.</a> Journal of Visualized Experiments. (149), e59499 (2019).</li><li>Jeffery, E., Church, C. D., Holtrup, B., Colman, L., Rodeheffer, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+depot-specific+activation+of+adipocyte+precursor+cells+at+the+onset+of+obesity.">Rapid depot-specific activation of adipocyte precursor cells at the onset of obesity.</a> Nature Cell Biology. 17 (4), 376-385 (2015).</li><li>Kim, S. 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=Loss+of+white+adipose+hyperplastic+potential+is+associated+with+enhanced+susceptibility+to+insulin+resistance.">Loss of white adipose hyperplastic potential is associated with enhanced susceptibility to insulin resistance.</a> Cell Metabolism. 20 (6), 1049-1058 (2014).</li><li>Vishvanath, 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=Pdgfrbeta++Mural+Preadipocytes+Contribute+to+Adipocyte+Hyperplasia+Induced+by+High-Fat-Diet+Feeding+and+Prolonged+Cold+Exposure+in+Adult+Mice.">Pdgfrbeta+ Mural Preadipocytes Contribute to Adipocyte Hyperplasia Induced by High-Fat-Diet Feeding and Prolonged Cold Exposure in Adult Mice.</a> Cell Metabolism. 23 (2), 350-359 (2016).</li><li>Wang, Q. A., Tao, C., Gupta, R. K., Scherer, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+adipogenesis+during+white+adipose+tissue+development,+expansion+and+regeneration.">Tracking adipogenesis during white adipose tissue development, expansion and regeneration.</a> Nature Medicine. 19 (10), 1338-1344 (2013).</li><li>Gao, Z., Daquinag, A. C., Su, F., Snyder, B., Kolonin, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDGFRalpha/PDGFRbeta+signaling+balance+modulates+progenitor+cell+differentiation+into+white+and+beige+adipocytes.">PDGFRalpha/PDGFRbeta signaling balance modulates progenitor cell differentiation into white and beige adipocytes.</a> Development. 145 (1), 155861 (2018).</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><li>Church, C. D., Berry, R., Rodeheffer, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+study+of+adipocyte+precursors.">Isolation and study of adipocyte precursors.</a> Methods Enzymol. 537, 31-46 (2014).</li><li>Buffolo, 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=Identification+of+a+Paracrine+Signaling+Mechanism+Linking+CD34(high)+Progenitors+to+the+Regulation+of+Visceral+Fat+Expansion+and+Remodeling.">Identification of a Paracrine Signaling Mechanism Linking CD34(high) Progenitors to the Regulation of Visceral Fat Expansion and Remodeling.</a> Cell Reports. 29 (2), 270-282 (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=De+novo+adipocyte+differentiation+from+Pdgfrbeta(+)+preadipocytes+protects+against+pathologic+visceral+adipose+expansion+in+obesity.">De novo adipocyte differentiation from Pdgfrbeta(+) preadipocytes protects against pathologic visceral adipose expansion in obesity.</a> Nature Communications. 9 (1), 890 (2018).</li><li>Lee, P. Y., Wang, J. X., Parisini, E., Dascher, C. C., Nigrovic, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ly6+family+proteins+in+neutrophil+biology.">Ly6 family proteins in neutrophil biology.</a> Journal of Leukocyte Biology. 94 (4), 585-594 (2013).</li><li>Vijay, 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=Single-cell+analysis+of+human+adipose+tissue+identifies+depot+and+disease+specific+cell+types.">Single-cell analysis of human adipose tissue identifies depot and disease specific cell types.</a> Nature Metabolism. 2 (1), 97-109 (2020).</li></ol>

T.A.P.は、ノボ ノルディスクA/Sの従業員であり、株主です

マウスのC57BL/6系統は、食事誘発性肥満の研究で最も使用されているマウス系統です。C57BL/6マウスは、高脂肪食(HFD)を摂取すると急速に体重が増加し、肥満に関連するメタボリックシンドロームの顕著な特徴(インスリン抵抗性や高脂血症など)を発現します。特に、高脂肪食(HFD)の摂食に関連して起こるWATの拡大は、デポ特異的な方法で起こる19,20,21,22,23。<sup class="xref...

白色脂肪組織(WAT)は、哺乳類のエネルギー貯蔵の主要な部位を表しています。この組織内では、脂肪細胞、または「脂肪細胞」が過剰なカロリーをトリグリセリドの形で貯蔵し、大きな単房性脂肪滴にパッケージ化されています。さらに、脂肪細胞は、エネルギー恒常性の様々な側面を調節する多数の因子を分泌する1,2,3。脂肪細胞はWATボリュームの大部分を占めています。ただし、脂肪細胞はWAT 4,5に見られる全細胞の50%未満しか占めていません。WATの非脂肪細胞コンパートメント、または間質血管画分(SVF)は、非常に不均一であり、血管内皮細胞、組織常在免疫細胞、線維芽細胞、および脂肪細胞前駆細胞(APC)集団が含まれています。
WATは、エネルギー貯蔵の需要の増加に伴ってサイズを拡大するという驚くべき能力において、非常に優れています。WATでの脂質の適切な保存は、非脂肪組織への有害な異所性脂質沈着から保護するため、この組織の可塑性を維持することが不可欠です6。カロリー過剰に反応して個々のWATデポがこの拡大を経験する方法は、肥満の状況におけるインスリン感受性の重要な決定要因です7。メタボリックシンドロームの肥満患者に観察される病理学的WAT拡張は、代謝的に好ましい皮下脂肪組織を犠牲にして、内臓WATデポの優先的な拡大によって特徴付けられます。さらに、肥満におけるインスリン抵抗性は、WATの病理学的リモデリングに関連しています。これは、既存の脂肪細胞の肥大成長(サイズの増加)、不十分な血管新生、慢性代謝炎症、細胞外マトリックス成分の蓄積(線維化)、および組織の低酸素症によって特徴付けられます8,9。これらの肥満のWAT表現型は、脂肪異栄養症(機能的WATの欠如)の状態で観察されるものと同様に、脂肪肝およびインスリン抵抗性と関連しています。対照的に、健康なWATの拡大は代謝的に健康な肥満集団で観察され、脂肪細胞過形成10を介した保護皮下WATおよびデポー拡大の優先的な拡大によって特徴付けられます。新しい脂肪細胞の動員は、脂肪細胞前駆細胞(APC)からのde novo脂肪細胞の分化(「脂肪形成」と呼ばれる)によって媒介されます。脂肪細胞の過形成は、比較的低い程度のWAT線維症および代謝性炎症と一致します6,11。WAT微小環境内の多数の細胞型は、肥満におけるWATの健康と拡張性に直接影響します12。そのため、WATに存在するさまざまな細胞タイプの機能を定義することは、この分野にとって依然として高い優先事項です。
過去10年間で、ヒトおよびマウスのWAT SVF13から天然APCを定義し、単離するためにいくつかの戦略が採用されてきました。このような戦略では、抗体ベースの細胞分離技術を用いて、一般的な間葉系幹細胞/前駆細胞マーカーの細胞表面発現に基づいてAPCを単離します。これらのアプローチには、蛍光標識抗体を用いた蛍光活性化細胞選別(FACS)や、免疫磁気ビーズ分離(すなわち、化学的に修飾された抗体)が含まれます。APCの単離を標的とする細胞表面タンパク質には、PDGFRα、PDGFRβ、CD34、およびSCA-1が含まれます。これらのアプローチは、APCの充実に役立っています。しかし、これらのマーカーに基づいて単離された細胞集団は、かなり不均一です。ごく最近のシングルセルRNAシーケンシング(scRNA-seq)研究では、マウスWAT 14,15,16,17の単離された間質血管画分(SVF)内の間質細胞の分子的および機能的不均一性が浮き彫りになっています。私たち自身のscRNA-seqおよび機能解析から、成体マウスの腹腔内WATの間質コンパートメントにおける機能的に異なる免疫調節および脂肪形成PDGFRβ+血管周囲細胞亜集団を同定し、特徴付けました15。線維炎症性前駆体(FIP)は、PDGFRβ+細胞の顕著な亜集団であり、LY6C発現(LY6C+ PDGFRβ+細胞)に基づいて単離することができる15。FIPは脂肪形成能力を欠き、さまざまな刺激に対して強い炎症誘発性反応を発揮し、コラーゲンを生成し、抗脂肪生成因子を分泌します15。これらの細胞の炎症誘発性および線維形成活性は、マウスの肥満に関連して増加し、これらの細胞がWATリモデリングの調節因子として関与しています。LY6C- CD9- PDGFRβ+亜集団は、脂肪細胞前駆細胞(APC)を表します。これらのAPCは、Ppargおよび他の脂肪形成促進遺伝子の発現に富んでおり、in vitroおよびin vivoで成熟脂肪細胞に容易に分化します15。ここでは、FACSを使用して成体マウスの腹腔内WATデポからこれらの異なる細胞集団を単離し、ビオチン化抗体を使用して免疫磁気ビーズを分離するための詳細なプロトコルを提供します。このプロトコルは、成体雄および雌マウスの複数の腹腔内WATデポから機能的に異なる脂肪前駆細胞亜集団を単離するために使用することができる15。これらの機能的に異なる細胞集団を単独で研究することは、健康と疾患における脂肪形成と腹腔内脂肪組織のリモデリングを調節する分子メカニズムの現在の理解に大きく貢献する可能性があります。
以下のプロトコルは、マウス精巣上体WATからの脂肪前駆細胞の単離を詳述しています。しかし、同じ手順を使用して、雄および雌マウス15の両方の腸間膜および後腹膜WATデポから対応する細胞を単離することができる。マウスでこれらのデポを同定し、単離する方法に関する詳細なプロトコルは、Bagchi et al.18に記載されています。このプロトコルは、6〜8週齢のマウスの使用に最適化されています。APCの頻度と分化能力は、加齢に伴って低下する可能性があります。

<table>
	<tbody>
		<tr>
			<td>Mechanical Tissue Preparation and SVF Isolation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>40 and 100 &#181;m cell strainers</td>
			<td>Fisher Scientific</td>
			<td>352340/352360</td>
			<td></td>
		</tr>
		<tr>
			<td>1X Phosphate buffered saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>21040CV</td>
			<td></td>
		</tr>
		<tr>
			<td>5ml polypropylene tubes</td>
			<td>Fisher Scientific</td>
			<td>352053</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digestion Buffer (for 10mL)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml HBSS</td>
			<td>Sigma</td>
			<td>H8264</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mg Collagenase D (1 mg/ml final cc.)</td>
			<td>Roche</td>
			<td>11088882001</td>
			<td></td>
		</tr>
		<tr>
			<td>0.15 g BSA (1.5 % final cc.)</td>
			<td>Fisher Scientific</td>
			<td>BP1605-100</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Immunomagnetic separation of APCs and non-APCs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5X MojoSort Buffer (MS buffer)</td>
			<td>BioLegend</td>
			<td>480017</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml MojoSort Magnet (MS magnet)</td>
			<td>BioLegend</td>
			<td>480019</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;L MojoSort Streptavidin Nanobeads</td>
			<td>BioLegend</td>
			<td>480015</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Purity Check and FACS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10X Red Blood Cell Lysis Buffer</td>
			<td>eBioscience</td>
			<td>00-4300-54</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc block (Mouse CD16/CD32)</td>
			<td>eBioscience</td>
			<td>553141</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin CD45</td>
			<td>BioLegend</td>
			<td>103103</td>
			<td>Concentration: &#8804; 0.25 &#181;g per 10^6 cells 
			Species: Mouse 
			Clone: 30-F11</td>
		</tr>
		<tr>
			<td>Biotin CD31</td>
			<td>BioLegend</td>
			<td>102503</td>
			<td>Concentration: &#8804; 0.25 &#181;g per 10^6 cells 
			Species: Mouse 
			Clone: MEC13.3</td>
		</tr>
		<tr>
			<td>Biotin CD9</td>
			<td>BioLegend</td>
			<td>124803</td>
			<td>Concentration: &#8804; 0.25 &#181;g per 10^6 cells 
			Species: Mouse 
			Clone: MZ3</td>
		</tr>
		<tr>
			<td>Biotin LY6C</td>
			<td>BioLegend</td>
			<td>128003</td>
			<td>Concentration: &#8804; 0.25 &#181;g per 10^6 cells 
			Species: Mouse 
			Clone: HK1.4</td>
		</tr>
		<tr>
			<td>CD31-PerCP/Cy5.5</td>
			<td>BioLegend</td>
			<td>102419</td>
			<td>Concentration: Dilution 1:400 
			Species: Mouse 
			Clone: 390</td>
		</tr>
		<tr>
			<td>CD45-PerCP/Cy5.5</td>
			<td>BioLegend</td>
			<td>103131</td>
			<td>Concentration: Dilution 1:400 
			Species: Mouse 
			Clone: 30-F11</td>
		</tr>
		<tr>
			<td>CD140b PDGFR&#946;-PE</td>
			<td>BioLegend</td>
			<td>136006</td>
			<td>Concentration: Dilution 1:50 
			Species: Mouse 
			Clone: APB5</td>
		</tr>
		<tr>
			<td>LY6C-APC</td>
			<td>BioLegend</td>
			<td>128016</td>
			<td>Concentration: Dilution 1:400 
			Species: Mouse 
			Clone: HK1.4</td>
		</tr>
		<tr>
			<td>CD9-FITC</td>
			<td>BioLegend</td>
			<td>124808</td>
			<td>Concentration: Dilution 1:400 
			Species: Mouse 
			Clone: MZ3</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture and Differentiation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gonadal APC Culture media (for 500mL)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>288 mL DMEM with 1 g/L glucose</td>
			<td>Corning</td>
			<td>10-014-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>192 mL MCDB201</td>
			<td>Sigma</td>
			<td>M6770</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Fetal bovine serum (FBS)** lot#14E024</td>
			<td>Sigma</td>
			<td>12303C</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL 100% ITS premix</td>
			<td>BD Bioscience</td>
			<td>354352</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL 10 mM L-ascorbic acid-2-2phosphate</td>
			<td>Sigma</td>
			<td>A8960-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>50 &#181;L 100 g/ml FGF-basic</td>
			<td>R&#38;D systems</td>
			<td>3139-FB-025/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Pen/Strep</td>
			<td>Corning</td>
			<td>30-001-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>500 &#181;L Gentamycin</td>
			<td>Gibco</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr>
			<td>**NOTE: The adipogenic capacity of primary APCs can vary from lot to lot of commercial FBS. Multiple lots/sources of FBS should be tested.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal adipose depots

白色脂肪組織(WAT)の間質血管画分(SVF)は、非常に不均一であり、成人期のWATの拡張とリモデリングに機能的に寄与する多数の細胞タイプで構成されています。この細胞の不均一性の影響を研究する上での大きな障壁は、in vitroおよびin vivo分析のためにWAT SVFから機能的に異なる細胞亜集団を容易に分離できないことです。シングルセルシーケンシング技術は最近、成体マウスの腹腔内WATデポにおいて、機能的に異なる線維炎症性および脂肪形成性PDGFRβ+血管周囲細胞亜集団を同定しました。線維炎症性前駆細胞(「FIP」と呼ばれる)は、炎症誘発性の表現型を発揮できる非脂肪原性コラーゲン産生細胞です。PDGFRβ+脂肪細胞前駆細胞(APC)は、細胞移植時にin vitroおよびin vivoの両方で高い脂肪形成を示します。ここでは、マウスの腹腔内WATデポからこれらの間質細胞亜集団を単離するための複数の方法について説明します。FIPおよびAPCは、蛍光活性化セルソーティング(FACS)またはビオチン化抗体ベースの免疫磁気ビーズ技術を利用することにより、単離できます。単離された細胞は、分子解析や機能解析に利用することができます。間質細胞亜集団の機能特性を単独で研究することで、細胞レベルでの生理学的または病理学的条件下での脂肪組織リモデリングに関する現在の知識が広がります。

このプロトコルは、成体マウスの腹腔内WATデポから異なる間質細胞集団の単離を可能にする2つの戦略を説明しています。APCおよびFIPは、FACS(図1)またはビオチン化抗体による免疫磁気ビーズ分離(図2)によって単離できます。どちらのアプローチも、すべて市販の試薬と抗体を利用します。免疫磁気ビーズの分離は、gWAT SVFからの脂肪形成細胞と非脂...

Read this Scientific Article Protocol about Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots at JoVE.com

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

このプロトコルは、蛍光活性化細胞選別または免疫磁気ビーズ分離により、マウス腹腔内白色脂肪組織(WAT)デポから脂肪形成および線維炎症性間質細胞亜集団を単離するための技術的アプローチを説明しています。

マウス腹腔内脂肪貯蔵所からの脂肪形成性および線維炎症性間質細胞亜集団の単離

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

isolation-adipogenic-fibro-inflammatory-stromal-cell-subpopulations

Research

JoVE Journal

Immunology and Infection

2.5K ビュー.  Novo Nordisk A/S. このプロトコルは、蛍光活性化細胞選別または免疫磁気ビーズ分離により、マウス腹腔内白色脂肪組織(WAT)デポから脂肪形成および線維炎症性間質細胞亜集団を単離するための技術的アプローチを説明しています。

ビデオ: Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

臍帯血から前駆B細胞サブセットの分離

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

免疫・感染学

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

脂肪組織の免疫細胞の単離

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

マウス新生児から保護免疫表現と機能皮質ミクログリアの分離

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

ネズミリンパ節間質細胞の単離

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

母体·胎児界面におけるマウス組織からの白血球の分離

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

単離とマウスリンパ球の活性化

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

分離、拡張、および CD31 cd34 陽性 + + ひと大網と皮下脂肪組織から血管内皮細胞の脂肪分化誘導

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development and autoimmune diseases. This is facilitated through a process of capture from free flow onto the luminal surface of activated endothelial cells, followed by their adhesion and transendothelial migration (transmigration) into the underlying affected tissue. However, the mechanisms that support the preferential and context-dependent recruitment of monocyte subpopulations are still not fully understood. Therefore, we have developed a method that allows the recruitment of different monocyte subpopulations to be simultaneously visualized and measured under flow. This method, based on time-lapse confocal imaging, allows for the unambiguous distinction between adherent and transmigrated monocytes. Here, we describe how this method can be used to simultaneously study the recruitment cascade of pro-angiogenic and non-angiogenic monocytes in vitro. Furthermore, this method can be extended to study the different steps of recruitment of up to three monocyte populations.

Here, we present an integrated protocol that measures monocyte subpopulation trafficking under flow in vitro by use of specific surface markers and confocal fluorescence microscopy. This protocol can be used to explore sequential recruitment steps as well as to profile other leukocyte subtypes using other specific surface markers.

体外流れ球サブポピュレーションの募集の同時研究

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

下流の単一細胞分析のため尿路感染症のマウスモデルから生成された単一細胞内細菌群集の分離

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

コラゲナーゼEを用いてマウスコロンからのラミナプロプリア単核細胞の単核細胞の単核細胞の単核細胞の単核細胞の単核細胞の単核細胞の単核細胞の単核細胞の単核細胞の単核細胞

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...