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>

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Yazarların açıklayacak bir şeyi yok.

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 border="0" cellpadding="0" cellspacing="0" style="width:799px;" width="799">
	<colgroup>
		<col />
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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">1.7 mL microcentrifuge tube</td>
			<td style="width:181px;">VWR</td>
			<td style="width:119px;">87003-294</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">13 mL culture tube</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">50-809-216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">15 mL conical tube</td>
			<td style="width:181px;">Greiner Bio-one</td>
			<td style="width:119px;">188 271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">5 mL test tube with cell strainer snap cap</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">08-771-23</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">50 mL conical tube</td>
			<td style="width:181px;">Greiner Bio-one</td>
			<td style="width:119px;">227 261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">70 &#181;m cell strainer</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">22-363-548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti-CD31-FITC antibody</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-102-519</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti-CD45-FITC antibody</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-102-491</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti-Sca1-APC antibody</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-102-833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti-Ter119-FITC antibody</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-112-908</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">BSA</td>
			<td style="width:181px;">Gold Biotechnology</td>
			<td style="width:119px;">A-420-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Collagenase type II</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">17101-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">DAPI</td>
			<td style="width:181px;">Thermo Fisher</td>
			<td style="width:119px;">D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">14190-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">F-12 medium</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11765-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">FcR blocking reagent</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-092-575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Hanks&#39; balanced salt solution (HBSS)</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">14025-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Horse serum</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">16050-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Penicillin-streptomycin</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Propidium iodide solution</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">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 ...

preparation-adipose-progenitor-cells-from-mouse-epididymal-adipose

Research

JoVE Journal

Biology

5.5K 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 Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

İsteyerek Pluripotent Kök Hücrelerinin Elde Edilen Fare Üretimi

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Biyoloji

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

İnsan Lipoaspirates den Yağ-türetilen Kök Hücre Manuel İzolasyon

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic lineages. Adipogenic differentiation of progenitor cells is a major mechanism driving adipose tissue expansion and dysfunction in response to obesity. Understanding changes to perivascular adipose tissue (PVAT) is thus clinically relevant in metabolic disease. However, previous studies have been predominately performed in the mouse and other animal models. This protocol uses human thoracic PVAT samples collected from patients undergoing coronary artery bypass graft surgery. Adipose tissue from the ascending aorta was collected and used for explantation of the stromal vascular fraction. We previously confirmed the presence of adipose progenitor cells in human PVAT with the capacity to differentiate into lipid-containing adipocytes. In this study, we further analyzed the differentiation potential of cells from the stromal vascular fraction, presumably containing multi-potent progenitor cells. We compared PVAT-derived cells to human bone marrow MSC for differentiation into adipogenic, osteogenic, and chondrogenic lineages. Following 14 days of differentiation, specific stains were utilized to detect lipid accumulation in adipocytes (Oil red O), calcific deposits in osteogenic cells (Alizarin Red), or glycosaminoglycans and collagen in chondrogenic cells (Masson&#8217;s Trichrome). While bone marrow MSC efficiently differentiated into all three lineages, PVAT-derived cells had adipogenic and chondrogenic potential, but lacked robust osteogenic potential.

The goal of this protocol is to test the ability of progenitor cells derived from human perivascular adipose tissue to differentiate into multiple cell lineages. Differentiation was compared to mesenchymal stem cells derived from human bone marrow, which is known to differentiate into adipocyte, osteocyte, and chondrocyte lineages.

İnsan aort Perivasküler yağ progenitör hücre farklılaşması kapasitesi

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic ...

Identification and Dissection of Diverse Mouse Adipose Depots

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, uncoupling of metabolism to generate heat, and secretion of adipokines to regulate whole-body homeostasis and immune responses. Emerging research is identifying important regional differences in the developmental, molecular, and functional profiles of adipocytes located in discrete depots throughout the body. Different properties of the depots are medically relevant since metabolic diseases often demonstrate depot-specific effects. This protocol will provide investigators with a detailed anatomic atlas and dissection guide for the reproducible and accurate identification and excision of diverse mouse adipose tissues. Standardized dissection of discrete adipose depots will allow detailed comparisons of their molecular and metabolic characteristics and contributions to local and systemic pathologic states under various nutritional and environmental conditions.

Adipocytes exist in discrete depots and have diverse roles within their unique microenvironments. As regional differences in adipocyte character and function are uncovered, standardized identification and isolation of depots is crucial for advancement of the field. Herein, we present a detailed protocol for the excision of various mouse adipose depots.

Çeşitli fare Adipose depoları tanımlama ve diseksiyon

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

Perivasküler Adipose dokularında modülasyon odaklı Izole mezenterik arterlerin kullanıldığı vasküler ton tepkisini değerlendirmek

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic ...

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity is characterized by an increase in adipose tissue (AT) mass due to adipocyte hyperplasia and/or hypertrophia, leading to profound remodeling of its three-dimensional structure. Indeed, the maximal capacity of AT to expand during obesity is pivotal to the development of obesity-associated pathologies. This AT expansion is an important homeostatic mechanism to enable adaptation to an excess of energy intake and to avoid deleterious lipid spillover to other metabolic organs, such as muscle and liver. Therefore, understanding the structural remodeling that leads to the failure of AT expansion is a fundamental question with high clinical applicability. In this article, we describe a simple and fast clearing method that is routinely used in our laboratory to explore the morphology of mouse and human white adipose tissue by fluorescent imaging. This optimized AT clearing method is easily performed in any standard laboratory equipped with a chemical hood, a temperature-controlled orbital shaker and a fluorescent microscope. Moreover, the chemical compounds used are readily available. Importantly, this method allows one to resolve the 3D AT structure by staining various markers to specifically visualize the adipocytes, the neuronal and vascular networks, and the innate and adaptive immune cells distribution.

Here, we describe a simple, inexpensive and fast clearing method to resolve the 3D structure of both mouse and human white adipose tissue using a combination of markers to visualize vasculature, nuclei, immune cells, neurons, and lipid-droplet coat proteins by fluorescent imaging.

Metiyatisilin Temizleme ve 3D Görüntüleme ile Yağ Dokusu Yapısının Araştırılması

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity ...

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4&#8211;9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14&#8211;21 days of differentiation using a cocktail of 0.5 &#956;g/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 &#956;M indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.

In this article, we describe isolation of rhesus macaque derived adipose-derived stem cells (ADSCs) using an enzymatic tissue digestion protocol. Next, we describe ADSC proliferation which includes cell detachment, counting and plating. Lastly, ADSC differentiation is described using specific adipogenic inducing agents. Additionally, we describe staining techniques to confirm differentiation.

Rhesus Macaque Adipoz Türevi Kök Hücrelerin İzolasyonu, Proliferasyonu ve Farklılaşması

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) ...

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods are essential to provide critical insights into these thermogenic adipose depots. Here, the protocol presents a PET/MR imaging-based method to evaluate the activity of brown and beige adipocytes in mouse interscapular brown adipose tissue (iBAT) and inguinal subcutaneous white adipose tissue (iWAT). Visualization and quantification of the thermogenic adipose depots were achieved using [18F]FDG, the non-metabolizable glucose analog, as the radiotracer, when combined with the precise anatomical information provided by MR imaging. The PET/MR imaging was conducted 7 days after cold acclimation and quantitation of [18F]FDG signal in different adipose depots was conducted to assess the relative mobilization of thermogenic adipose tissues. Removal of iBAT substantially increased cold-evoked [18F]FDG uptake in iWAT of the mice.

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Functional imaging and quantitation of thermogenic adipose depots in mice using a micro-PET/MR imaging-based approach.

[18F]FDG Micro-PET/MR Görüntüleme Kullanılarak Farelerde Kahverengi ve Bej Yağ Dokularının Görselleştirilmesi ve Ölçülmesi

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods ...

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

Vasküler Endotel Hücrelerinin Aşağı Akış Uygulamaları için Farklı Yağ Depolarından İzolasyonu ve Tanımlanması

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...

Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with multiple lipid droplets, a central nucleus, a high mitochondrial content, and the expression of uncoupling protein 1 (UCP1). BAT has been proposed as a potential therapeutic target for obesity and its associated metabolic disorders due to its ability to dissipate metabolic energy as heat. To investigate BAT function and regulation, brown adipocyte culturing is indispensable. The present protocol optimizes tissue processing and cell differentiation for culturing brown adipocytes from newborn mice. Additionally, procedures for the imaging of differentiated adipocytes with both confocal immunofluorescence and transmission electron microscopy are shown. In the brown adipocytes differentiated with the techniques described herein, the major defining features of classical BAT are preserved, including high UCP1 levels, increased mitochondrial mass, and very close physical contact between the lipid droplets and mitochondria, making this method a valuable tool for BAT studies.

Preadipocytes are isolated from the stromal vascular fraction of interscapular brown adipose tissue from newborn mice and differentiated into cells that accumulate lipid droplets, express molecular markers, and show the mitochondrial morphology of mature brown adipocytes. These cells are further analyzed by immunofluorescence and transmission electron microscopy.

Yenidoğan Farelerden İnterskapular Yağ Dokusunun Stromal Vasküler Fraksiyonundan Kahverengi Adipositlerin Farklılaşması ve Görüntülenmesi

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with ...