Name: preparation of adipose progenitor cells from mouse epididymal adipose tissues
Uploaded: 2020-08-25T14:45:00.000+00:00
Duration: 6 min 17 s
Description: Watch this Scientific Journal Video about Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues at JoVE.com

FACS sıralama konusunda yardım için Mayo Clinic Mikroskopi Hücre Analizi Çekirdek Akış Sitometri Tesisi'ni kabul ediyoruz.

Tüm hayvan deneysel işlemleri Mayo Clinic Kurumsal Hayvan Bakım ve Kullanım Komitesi tarafından onaylanarak gerçekleştirildi.
1. Çözelti hazırlama 
<ol><li>Kollajenaz II 2 g'ı 100 mL Hanks'in dengeli tuz çözeltisinde (HBSS) çözerek kollajenaz% 2 (w/v) çözeltisi hazırlayın. Her kullanım için aliquot 200 μL her biri.</li>
	<li>84 mL F-12 orta, 15 mL at serumu ve 1 mL penisilin/streptomisisini karıştırarak nötralizasyon ortamını hazırlayın.</li>
	<li>Dulbecco'nun fosfat tamponlu salininin (DPBS) 100 mL'sinde 500 mg sığır serum albümini (BSA) ve 400 μL 0,5 M EDTA'yı eriterek akış sitometri tamponu hazırlayın.</li>
</ol>2. Epididimal yağ yastığının diseksiyonu ve doku ayrışması
<ol><li>İnsanca ötenazi (izofluran/servikal çıkık) bir erkek fare. Bu protokolde dört aylık bir FVB fare kullanıldı.</li>
	<li>Karın bölgesine %70 etanol uygulayın/püskürtün ve temiz makas ve önps kullanarak alt karın boşluğunu ortaya çıkarın.</li>
	<li>Epididimal yağ yastığını bulun (proksimal/testislere bağlı). Testisler ve epididimal yağ pedi arasındaki kavşağı künt toparlamalarla tutun ve epididimal yağ yastığını serbest bırakmak için hafifçe çekin.</li>
	<li>Makas kullanarak keserek testisleri çıkarın ve 15 dakika boyunca oda sıcaklığında 50 mL konik bir tüpte% 3 BSA ile desteklenmiş 5 mL HBSS'de epididimal yağ yastığını kuluçkaya yatırın.</li>
	<li>4 °C'de 7 dakika boyunca 150 x g'da santrifüj.</li>
	<li>50 mL konik tüpten yüzen epididimal yağ yastığı alın ve temiz makas kullanarak dokuyu ince bir şekilde kıyın.</li>
	<li>13 mL kültür tüpünde 3,8 mL HBSS'ye 200 μL kollajenaz% 2 çözelti ekleyin. Kıyılmış dokuyu ekleyin ve 37 °C'de 1 saat boyunca 5 rpm'de dönen bir inkübatörde kuluçkaya yatırın. Dönen bir inkübatör yerine, 37 °C'deki hücre kültürü inkübatörü alternatif olarak zaman zaman ajitasyonlarla kullanılabilir.</li>
	<li>Tüm içeriği 50 mL konik bir tüpe aktarın ve 10 mL nötralizasyon ortamı ekleyin. Tüpü 2-3 kez ters çevirerek hafifçe karıştırın.</li>
	<li>70 μm hücre süzgeç ile donatılmış başka bir 50 mL konik tüp hazırlayın. Hücre süzgecini kullanarak sindirilen dokuyu yeni tüpe filtreleyin.</li>
	<li>Akışı 4 °C'de 10 dakika boyunca 350 x g'da 15 mL konik tüpe ve santrifüje aktarın.</li>
	<li>5 mL DPBS ile süpernatant ve resuspend hücre peletlerini dikkatlice çıkarın. 4 °C'de 10 dakika boyunca 350 x g'da santrifüj.</li>
	<li>Süpernatantı dikkatlice çıkarın ve 50 μL akış sitometri tamponu ekleyin. Hafifçe pipetleyarak iyice karıştırın ve hücreleri buzda tutun. Hücre peletlerinin hacmi ve az miktarda artık süpernatant nedeniyle, akış sitometri tamponundaki hücrelerin toplam hacmi 50 μL'den (yaklaşık 100 μL) büyük olacaktır.</li>
</ol>3. Antikor etiketleme ve floresan aktif hücre sıralama (FACS)
<ol><li>Hücreleri nazik pipetleme ile iyice karıştırdıktan sonra, hücre süspansiyonunun 54 μL'lik kısmını 1,7 mL'lik bir mikrosantrifüj tüpüne aktarın. 6 μL FcR blokaj reaktifi ekleyin ve hafifçe pipetleyarak iyice karıştırın. 4 °C'de 10 dakika kuluçkaya yatır. 54 μL aldıktan sonra kalan hücreleri kaydedin ve 3.5 ve adım 3.6'da yersiz bir kontrol olarak kullanmak için buzda tutun.</li>
	<li>3.1. adımdaki inkübasyon sırasında, anti-Sca1-APC, anti-Ter119-FITC, anti-CD31-FITC ve anti-CD45-FITC antikorlarının her birini bir mikrosantrifüj tüpünde birleştirerek bir antikor kokteyli hazırlayın. Hafif pipetleme ile iyice karıştırın ve ışıktan korunan buz üzerinde tutun.</li>
	<li>FcR bloke reaktifi ile 10 dakika inkübasyondan sonra, antikor kokteylinin 40 μL'lik kısmını ekleyin ve hafif pipetleme ile iyice karıştırın. 4 °C'de 10 dakika kuluçkaya yatır.</li>
	<li>Lekeli hücrelere 1 μg/mL DAPI ile desteklenmiş 500 μL akış sitometri tamponu ekleyin. Hücre süzgeç yapışma kapağına sahip 5 mL'lik bir test tüpü kullanarak nazik pipetleme ve filtre hücreleri ile iyice karıştırın. Hücreleri buzda tutun.</li>
	<li>3.1. adımda kalan hücrelere 500 μL akış sitometri tamponu ekleyin ve hafif pipetleme ile iyice karıştırın. Hücre süzgeç yapıştırıcı kapaklı 5 mL test tüpü kullanarak hücreleri filtreleyin. Bu hücreleri buzda tutun ve aşağıdaki adımda bozulmamış bir kontrol olarak kullanın.</li>
	<li>Lekeli hücreleri ve bozulmamış kontrolü FACS analiz veya sıralama cihazına götürün.<ol>
<li>Şekil 1'de gösterilen gating stratejileriyle APC+/FITC-/DAPI- popülasyonu tanımlayın ve yalıtın. Bu hücreler uygulanabilir Sca1+/Ter119-/CD31-/CD45- hücrelerini temsil eder. Enkaz ve hücre agregaları ileri (FSC) ve yan dağılım (SSC) çizimleri kullanılarak tükenür. Daha sonra, DAPI- nüfus kapılı, ardından APC+/ FITC- nüfus gating. Gating parametrelerinin ayarlanmasına yardımcı olmak için ellenmemiş denetimler kullanılmalıdır.</li>
		<li>Hücreleri 1,5 mL mikrosantrifüj tüpünde 500 μL akış sitometri tamponunda toplayın. İleriye doğru, kontaminasyonu en aza indirmek için tüm prosedürler steril koşullar altında yapılmalıdır. Bir fareden yaklaşık 50.000 – 100.000 hücre toplanır.</li>
	</ol>
</li>
	<li>Hücre canlılığını değerlendirmek için hemositometre kullanarak hücreleri sayın. Hücre agregaları daha fazla aşağı akış analizini engelleyebileceğinden, hücre agregalarının varlığı da bu agregaların minimum varlığını (<5% uygulanabilir hücre sayısının) sağlamak için değerlendirilir.</li>
</ol>

<ol>
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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, 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, 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, 103-108 (2018).</li><li>Raajendiran, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Metabolically+Distinct+Adipocyte+Progenitor+Cells+in+Human+Adipose+Tissues.">Identification of Metabolically Distinct Adipocyte Progenitor Cells in Human Adipose Tissues.</a> Cell Reports. 27, 1528-1540 (2019).</li><li>De Matteis, R., 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=In+vivo+physiological+transdifferentiation+of+adult+adipose+cells.">In vivo physiological transdifferentiation of adult adipose cells.</a> Stem Cells. 27, 2761-2768 (2009).</li><li>Hanamsagar, R., 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=An+optimized+workflow+for+single-cell+transcriptomics+and+repertoire+profiling+of+purified+lymphocytes+from+clinical+samples.">An optimized workflow for single-cell transcriptomics and repertoire profiling of purified lymphocytes from clinical samples.</a> Scientific Reports. 10, 2219 (2020).</li><li>Marinovic, M. 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Tek hücreli RNA dizilimi (scRNA-seq), tek hücre düzeyindeki çeşitli hücre popülasyonlarını aynı anda incelemek için güçlü bir araç olarak hızla çekiş kazanıyor. Numune hazırlama ve yüksek verim sıralaması ile ilişkili yüksek maliyetler nedeniyle, deneysel başarı olasılığını artırmak için hücresel girdileri (yüksek canlılık ve saflık) optimize etmek zorunludur. Bazı hücre hazırlama protokolleri, FACS sıralama14olmadan düşük dönüşlü yıkamalar ve sü...

Yağ dokusu enerji metabolizmasında önemli bir rol oynar. Fazla enerji lipitler şeklinde depolanır ve yağ dokusu beslenme durumuna ve enerjik talebe bağlı olarak önemli genişleme veya geri çekilme yeteneğine sahiptir. Yağ dokusunun genişlemesi adiposit boyutundaki (hipertrofi) ve/veya adiposit sayısındaki (hiperplazi) artıştan; ikinci süreç, yağ progenitör hücrelerinin çoğalması ve farklılaşması ile sıkı bir şekilde düzenlenir1,2. Obezite sırasında, yağ dokusu aşırı genişler ve hipoksi, iltihaplanma ve insülin direnci de dahil olmak üzere doku disfonksiyonu genellikle3,4gelişir. Bu komplikasyonlar hipertansiyon, diyabet, kardiyovasküler hastalıklar, inme ve kanser dahil olmak üzere birçok kronik hastalık için risk faktörleridir5. Bu nedenle, kontrolsüz yağ dokusu genişlemesini sınırlamak ve yağ dokusu patolojilerini azaltmak en önemli biyomedikal araştırma öncelikleridir. Yağ dokusu genişlemesi sırasında, yerleşik yağ dokusundan türetilmiş kök hücreler (ASC'ler) çoğalır ve sırayla preadipositlere (taahhüt edilen progenitör hücreler) ve daha sonra olgun adipositlere farklılaşır6. Son tek hücreli RNA dizileme (scRNA-seq) çalışmaları, bu yağ progenitör hücre (APC) popülasyonlarının (ASC'ler ve preadipositler) önemli moleküler ve fonksiyonel heterojenlik 7,8 ,9,10,11,12. Örneğin, ASC'ler daha az adipojenik farklılaşma kapasitesi gösterirken, preadipositler7'yekıyasla daha yüksek çoğalma ve genişletme yetenekleri de sergiler. ASC ve preadiposit popülasyonlarında daha fazla moleküler farklılıklar bildirilmektedir, ancak bu farklılıkların işlevsel ilgisi belirsizliğini korumaktadır7. Birlikte, bu veriler yağ progenitor hücre havuzunun karmaşıklığını vurgular ve bu kritik hücre popülasyonlarını daha iyi anlamak ve manipüle etmek için araçlar geliştirme ve standartlaştırma ihtiyacının altını çizer.
Bu protokol, tek hücreli çalışmalar (scRNA-dizileme) ve hücre kültürü de dahil olmak üzere hassas aşağı akış analizleri için uygun olan fare epidididimal yağ pedlerinden yüksek canlılık Sca1+ yağ progenitör hücre popülasyonlarının izolasyonunun ayrıntılarını içerir. Epididimal yağ pedlerinin izolasyonu ve ayrışması, izole APC'lerin uygulanabilirliğini artıran hafif değişikliklerle daha önce açıklandığı gibi7,13 olarak gerçekleştirildi. Kısacası, epididimal yağ pedlerinden ayrışmış hücreler, hem ASC'ler hem de preadipositler6,7ve diğer soy (Lin) belirteçleri için bir belirteçolanSca1'e karşı antikorlarla lekelenir: Ter119 (eritroid hücreler), CD31 (endotel hücreleri) ve CD45 (lökositler). Uygulanabilir Sca1+/Ter119-/CD31-/CD45-/DAPI- hücreler daha sonra floresan aktif hücre sıralama (FACS) ile sıralanır. Daha da önemlisi, bu protokol, ASC'ler ve preadipositler içinde fonksiyonel olarak heterojen subpopulasyonları tanımlayan yeni bir tek hücreli RNA dizileme çalışmasında bildirilen uygulanabilir Sca1+/ Lin- yağ progenitör hücrelerinin başarılı izolasyonu ve analizi ile doğrulanmıştır7.

<table><tbody><tr><td>1.7 mL microcentrifuge tube</td><td>VWR</td><td>87003-294</td><td></td></tr><tr><td>13 mL culture tube</td><td>Thermo Fisher Scientific</td><td>50-809-216</td><td></td></tr><tr><td>15 mL conical tube</td><td>Greiner Bio-one</td><td>188 271</td><td></td></tr><tr><td>5 mL test tube with cell strainer snap cap</td><td>Thermo Fisher Scientific</td><td>08-771-23</td><td></td></tr><tr><td>50 mL conical tube</td><td>Greiner Bio-one</td><td>227 261</td><td></td></tr><tr><td>70 &amp;micro;m cell strainer</td><td>Thermo Fisher Scientific</td><td>22-363-548</td><td></td></tr><tr><td>Anti-CD31-FITC antibody</td><td>Miltenyi Biotec</td><td>130-102-519</td><td></td></tr><tr><td>Anti-CD45-FITC antibody</td><td>Miltenyi Biotec</td><td>130-102-491</td><td></td></tr><tr><td>Anti-Sca1-APC antibody</td><td>Miltenyi Biotec</td><td>130-102-833</td><td></td></tr><tr><td>Anti-Ter119-FITC antibody</td><td>Miltenyi Biotec</td><td>130-112-908</td><td></td></tr><tr><td>BSA</td><td>Gold Biotechnology</td><td>A-420-500</td><td></td></tr><tr><td>Collagenase type II</td><td>Thermo Fisher Scientific</td><td>17101-015</td><td></td></tr><tr><td>DAPI</td><td>Thermo Fisher</td><td>D1306</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (DPBS)</td><td>Thermo Fisher Scientific</td><td>14190-144</td><td></td></tr><tr><td>F-12 medium</td><td>Thermo Fisher Scientific</td><td>11765-054</td><td></td></tr><tr><td>FcR blocking reagent</td><td>Miltenyi Biotec</td><td>130-092-575</td><td></td></tr><tr><td>Hanks&#039; balanced salt solution (HBSS)</td><td>Thermo Fisher Scientific</td><td>14025-092</td><td></td></tr><tr><td>Horse serum</td><td>Thermo Fisher Scientific</td><td>16050-122</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Thermo Fisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>Propidium iodide solution</td><td>Miltenyi Biotec</td><td>130-093-233</td><td></td></tr></tbody></table>

preparation of adipose progenitor cells from mouse epididymal adipose tissues

Obezite ve diyabet, kalp hastalığı ve kanser gibi metabolik bozuklukların tümü dramatik yağ dokusu tadilatı ile ilişkilidir. Doku bazlı yağ progenitör hücreler (APC' ler) yağ dokusu homeostazında önemli bir rol oynar ve doku patolojisine katkıda bulunabilir. Tek hücreli RNA dizilimi ve tek hücreli proteomik dahil olmak üzere tek hücreli analiz teknolojilerinin artan kullanımı, popülasyon veya doku çapındaki değişiklikler bağlamında bireysel hücre ifade değişikliklerinin benzeri görülmemiş bir şekilde çözülmesine izin vererek kök/progenitör hücre alanını dönüştürmektedir. Bu yazıda, fare epidimal yağ dokusunu parçalamak, tek yağ dokusundan türetilmiş hücreleri izole etmek ve uygulanabilir Sca1+/CD31-/CD45-/Ter119- APC'ler için zenginleştirmek için floresan aktif hücre sıralamasını (FACS) gerçekleştirmek için ayrıntılı protokoller sunuyoruz. Bu protokoller, araştırmacıların tek hücreli RNA dizilimi gibi aşağı akış analizleri için uygun yüksek kaliteli APC'ler hazırlamalarını sağlayacaktır.

Bu deneyde dört aylık erkek FVB fareleri kullanıldı. FSC/SSC arazileri kullanılarak enkaz ve çiftlerin dışlanmasından sonra, uygulanabilir hücreler (DAPI- popülasyon) kapılıydı, ardından APC+/ FITC- popülasyon seçimi (Şekil 1). DAPI, APC ve FITC kapıları, tespit edilmemiş kontrole göre çizildi. Gating stratejileri Şekil 1'de gösterilmiştir.
1 saat sıralamadan sonra, izolasyon...

Watch this Scientific Journal Video about Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues at JoVE.com

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Floresan aktif hücre sıralamasını kullanarak fare epidididimal yağ pedlerinden yüksek oranda uygulanabilir yağ progenitör hücrelerini izole etmek için basit bir yöntem sunuyoruz.

Fare Epidididimal Yağ Dokularından Yağ Progenitör Hücrelerinin Hazırlanması

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

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JoVE Journal

Biology

5.6K Görüntüleme.  Mayo Clinic. Floresan aktif hücre sıralamasını kullanarak fare epidididimal yağ pedlerinden yüksek oranda uygulanabilir yağ progenitör hücrelerini izole etmek için basit bir yöntem sunuyoruz.

Video: Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

Tavan Kültürlerde Olgun adipositlerinde ve dediferansiyonunu ile İnsan adipoz Kök Hücre Üretimi

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

Gelişim Biyolojisi

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

Domuz Subkutan yağ dokularındaki İzolasyonu ve Yağ-Türetilmiş Kök Hücre Farklılaşması

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Isolation and Processing of Murine White Adipocytes for Transcriptome and Epigenome Analyses

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type in white adipose tissue. Understanding how adipocytes function and respond to (epi)genetic and environmental signals is essential for identifying the cause(s) of obesity. RNA and chromatin have previously been isolated from adipocytes using enzymatic digestion. In addition, protocols have been developed for nuclear isolation, where purification is achieved by fluorescence-activated cell sorting (FACS) of adipocyte-specific transgenic reporters. One of the greatest challenges to achieving high yield and quality during such protocols is the substantial amount of lipid contained in adipose tissue. The present protocol describes an optimized procedure for isolating mature adipocytes that leverages heptane to separate lipids from the targets of interest (RNA/chromatin). The resulting RNA has high integrity and generates high-quality RNA-seq results. Likewise, the procedure improves nuclei yield rate and generates reproducible ChIP-seq results across samples. Therefore, the current study provides a reliable and universal murine adipocyte isolation protocol suitable for whole-genome transcriptome and epigenome studies.

The present protocol summarizes a universal method for isolating, purifying, and upstream processing of murine white adipocytes optimized for downstream total RNA sequencing, Nuclei Extraction by SONication (NEXSON), and ChIP-seq.

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type ...

Genetik

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.

Yalıtım, genişleme ve Adipojenik indüksiyon CD34 + CD31 + insan Omental ve deri altı yağ dokusu endotel hücrelerden

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

İmmünoloji ve Enfeksiyon

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

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

Murin İntraabdominal Yağ Depolarından Adipojenik ve Fibroinflamatuar Stromal Hücre Alt Popülasyonlarının İzolasyonu

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

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

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

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

Primer Beyaz ve Kahverengi Preadipositlerin Yenidoğan Farelerden İzolasyonu ve Farklılaşması

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

Biyoloji

Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance contributes to the development of type 2 diabetes (T2D) and other obesity-derived diseases of high prevalence worldwide. Therefore, understanding the mechanisms underlying insulin resistance is of great relevance. Several models have been used to study insulin resistance both in vivo and in vitro; primary adipocytes represent an attractive option to study the mechanisms of insulin resistance and identify molecules that counteract this condition and the molecular targets of insulin-sensitizing drugs. Here, we have established an insulin resistance model using primary adipocytes in culture treated with tumor necrosis factor-&#945; (TNF-&#945;).
Adipocyte precursor cells (APCs), isolated from collagenase-digested mouse subcutaneous adipose tissue by magnetic cell separation technology, are differentiated into primary adipocytes. Insulin resistance is then induced by treatment with TNF-&#945;, a proinflammatory cytokine that reduces the tyrosine phosphorylation/activation of members of the insulin signaling cascade. Decreased phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS-1), and protein kinase B (AKT) are quantified by western blot. This method provides an excellent tool to study the mechanisms mediating insulin resistance in adipose tissue.

This protocol describes the isolation of mouse preadipocytes from subcutaneous fat, their differentiation into mature adipocytes, and the induction of insulin resistance. Insulin action is evaluated by the phosphorylation/activation of members of the insulin signaling pathway through western blot. This method allows direct determination of insulin resistance/sensitivity in primary adipocytes.

Primer Kültürde Diferansiye Fare Adipositleri: İnsülin Direncinin Bir Modeli

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance ...

Murin Perivasküler Yağ Dokusundan İzole Edilen Preadipositlerin Kültürlenmesi

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

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

Yazar Spotlight: Fare Perivasküler Yağ Dokusundan SVF'den Türetilmiş Preadipositlerin İzolasyonu, Kültürü ve Adipojenik İndüksiyonunun Önemi 

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

Fare Epidididimal Yağ Dokularından Yağ Progenitör Hücrelerinin Hazırlanması

Özet

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Tavan Kültürlerde Olgun adipositlerinde ve dediferansiyonunu ile İnsan adipoz Kök Hücre Üretimi

Domuz Subkutan yağ dokularındaki İzolasyonu ve Yağ-Türetilmiş Kök Hücre Farklılaşması

Isolation and Processing of Murine White Adipocytes for Transcriptome and Epigenome Analyses

Yalıtım, genişleme ve Adipojenik indüksiyon CD34 + CD31 + insan Omental ve deri altı yağ dokusu endotel hücrelerden

Murin İntraabdominal Yağ Depolarından Adipojenik ve Fibroinflamatuar Stromal Hücre Alt Popülasyonlarının İzolasyonu

Primer Beyaz ve Kahverengi Preadipositlerin Yenidoğan Farelerden İzolasyonu ve Farklılaşması

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