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

우리는 FACS 분류에 대한 지원을 위해 메이요 클리닉 현미경 세포 분석 코어 흐름 세포 분석 시설을 인정합니다.

모든 동물 실험 절차는 메이요 클리닉 기관 동물 관리 및 사용위원회의 승인을 받아 수행되었습니다.
1. 솔루션 준비 
<ol><li>콜라게나아제 2% (w/v) 용액을 100mL 행크스의 균형 잡힌 소금 용액(HBSS)으로 콜라게나아제 II 2g을 용해시켜 준비합니다. Aliquot 200 μL 각 사용에 대 한.</li>
	<li>84mL F-12 배지, 15mL 말 혈청, 1mL 페니실린/연쇄절제술을 혼합하여 중화 매체를 준비한다.</li>
	<li>덜베코의 인산완충식식염(DPBS)의 100mL에서 소 세럼 알부민(BSA)과 0.5M EDTA의 400 μL을 용해하여 유동 세포측정 버퍼를 준비한다.</li>
</ol>2. 부피성 지방 패드 및 조직 해리의 해부
<ol><li>인간적으로 안락사 (이소플루란 / 자궁 경부 탈구) 하나의 남성 마우스. 이 프로토콜에서는 4개월 된 FVB 마우스가 사용되었습니다.</li>
	<li>복부에 70% 에탄올을 바르고 깨끗한 가위와 집게를 사용하여 하복부를 노출시합니다.</li>
	<li>부피성 지방 패드(상반된 지방 패드/고환에 부착)를 찾습니다. 무딘 집게와 고환과 고환 지방 패드 사이의 접합을 잡고 부피 지방 패드를 해방부드럽게 당깁니다.</li>
	<li>실온에서 50mL 원피스 튜브에서 3%의 BSA로 보충된 HBSS5mL에서 가위를 사용하여 절단하여 고환을 제거하고 5mL 의 부피성 지방 패드를 15분 동안 제거합니다.</li>
	<li>원심분리기는 4°C에서 7분 동안 150 x g의 원심분리기.</li>
	<li>50mL 원뿔관에서 부동 부피 지방 패드를 가지고 깨끗한 가위를 사용하여 조직을 잘게 다진다.</li>
	<li>13mL 배양 튜브에 200 μL 콜라게나아제 2% 용액을 3.8mL의 HBSS에 추가합니다. 다진 조직을 추가하고 37 °C에서 1 시간 동안 5 rpm에서 회전 인큐베이터에 인큐베이션을 넣습니다. 회전 인큐베이터 대신 37°C의 세포 배양 인큐베이터는 가끔 동요와 함께 사용할 수 있습니다.</li>
	<li>전체 내용을 50mL 원문 튜브로 옮기고 10mL의 중화 매체를 추가합니다. 튜브를 2-3번 반전시켜 부드럽게 섞는다.</li>
	<li>70 μm 세포 스트레이너가 장착된 또 다른 50mL 원전 튜브를 준비합니다. 소화된 조직을 세포 여과기를 사용하여 새로운 튜브로 필터링합니다.</li>
	<li>4°C에서 10분 동안 350 x g에서 15mL 원심분리기및 원심분리기로 유동을 전달합니다.</li>
	<li>조심스럽게 상체를 제거하고 DPBS의 5 mL로 셀 펠릿을 다시 중단합니다. 4°C에서 10분 동안 350 x g의 원심분리기.</li>
	<li>상체를 조심스럽게 제거하고 50 μL의 유동 세포 측정 버퍼를 추가합니다. 부드럽게 파이펫으로 잘 섞어서 얼음 위에 세포를 보관하세요. 세포 펠릿의 부피와 소량의 잔류 상류체로 인해, 유동 세포 분석 버퍼내의 총 세포 부피는 50 μL(약 100 μL)을 초과할 것이다.</li>
</ol>3. 항체 라벨링 및 형광 활성화 셀 정렬(FACS)
<ol><li>세포를 부드러운 파이펫팅과 잘 혼합한 후 셀 서스펜션의 54 μL을 1.7mL 마이크로센심분리기 튜브로 옮기습니다. FcR 블로킹 시약 6μL을 추가하고 부드럽게 파이펫팅하여 잘 섞습니다. 4 °C에서 10 분 동안 배양하십시오. 54 μL을 복용한 후 남은 세포를 저장하고 얼음에 보관하여 3.5 단계 및 3.6 단계에서 스테인드 컨트롤로 사용하십시오.</li>
	<li>3.1 단계에서 의 배양 중에, 마이크로센티립후지 튜브에 각각 11 μL의 안티-Sca1-APC, 안티-테르119-FITC, 안티 CD31-FITC 및 안티 CD45-FITC 항체를 결합하여 항체 칵테일을 준비한다. 부드러운 파이펫팅으로 잘 섞어서 빛으로부터 보호되는 얼음을 유지하세요.</li>
	<li>FcR 블로킹 시약으로 10분 동안 배양한 후 항체 칵테일의 40 μL을 추가하고 부드러운 파이펫팅으로 잘 섞습니다. 4 °C에서 10 분 동안 배양하십시오.</li>
	<li>염색된 셀에 DAPI 1 μg/mL로 보충된 500 μL의 유동 세포분석 버퍼를 추가합니다. 셀 스트레이너 스냅 캡이 있는 5mL 테스트 튜브를 사용하여 부드러운 파이펫팅및 필터 셀로 잘 섞으세요. 얼음에 세포를 유지합니다.</li>
	<li>3.1 단계에서 남은 셀에 500 μL의 유동 세포 측정 버퍼를 추가하고 부드러운 파이펫팅으로 잘 섞습니다. 셀 스트레이너 스냅 캡이 있는 5mL 테스트 튜브를 사용하여 셀을 필터링합니다. 이 세포를 얼음위에 유지하고 다음 단계에서 스테인드 컨트롤로 사용하십시오.</li>
	<li>스테인드 셀과 스테인드 컨트롤을 FACS 분석 또는 정렬 기기로 가져 가라.<ol>
<li>도 1에표시된 게이팅 전략으로 APC+FITC-/DAPI- 인구를 식별하고 격리합니다. 이 세포는 실행 가능한 Sca1+/Ter119-/CD31-/CD45- 세포를 나타냅니다. 파편 및 셀 응집체는 전방(FSC) 및 측면 분산(SSC) 플롯을 사용하여 고갈됩니다. 그런 다음, DAPI- 인구가 문이 닫힌 다음 APC+/ FITC- 인구의 게이팅이 뒤따릅니다. 스테인드 컨트롤은 게이팅 매개 변수를 설정하는 데 사용되어야 합니다.</li>
		<li>1.5mL 마이크로센트심리후지 튜브에서 500μL의 유동 세포분석 완충제로 세포를 수집한다. 앞으로 모든 절차는 오염을 최소화하기 위해 멸균 조건에서 수행되어야합니다. 대략 50,000 - 100,000 세포는 1개의 마우스에서 집합됩니다.</li>
	</ol>
</li>
	<li>세포 생존가능성을 평가하기 위해 혈종계를 사용하여 세포를 계산합니다. 세포 집계는 추가 다운스트림 분석을 방해할 수 있기 때문에 셀 집계의 존재도 평가되어 이러한 응재의 최소 존재(실행 가능한 셀 수의 5%)를 보장합니다.</li>
</ol>

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저자는 공개 할 것이 없습니다.

단세포 RNA 시퀀싱(scRNA-seq)은 단일 세포 수준에서 다양한 세포 집단을 동시에 연구하는 강력한 도구로서 빠르게 견인력을 얻고 있다. 시료 준비와 높은 처리량 시퀀싱과 관련된 높은 비용으로 인해 실험 성공 가능성을 높이기 위해 세포 입력(높은 생존가능성 및 순도)을 최적화하는 것이 필수적입니다. 일부 세포 준비 프로토콜은 FACS 정렬14없이 낮은 스핀 세차및 컬럼 기반 분리?...

지방 조직은 에너지 대사에 중요한 역할을합니다. 과도한 에너지는 지질의 형태로 저장되며, 지방 조직은 영양 상태와 에너지 수요에 따라 상당한 확장 또는 후퇴가 가능합니다. 지방 조직의 확장은 지방 세포 크기 (비대) 및 /또는 지방 세포 수 (hyperhyperasia)의 증가에서 발생할 수 있습니다. 후자의 과정은 지방 선조 세포의 증식 및 분화에 의해 단단히조절된다 1,2. 비만 도중, 지방 조직은 과도하게 확장되고, 조직 기능 장애 – 저산소증을 포함하여, 염증 및 인슐린 저항 - 수시로3,4를개발합니다. 이러한 합병증은 고혈압, 당뇨병, 심혈관 질환, 뇌졸중 및 암5를포함한 많은 만성 질환에 대한 위험 요소입니다. 따라서 통제되지 않는 지방 조직 확장을 제한하고 지방 조직 병리학을 완화하는 것이 최고의 생체 의학 연구 우선 순위입니다. 지방 조직 팽창 동안, 상주 지방 조직 유래 줄기 세포 (ASC)는 증식하고 preadipocytes로 순차적으로 분화 (헌신적 인 전구 세포) 다음 성숙한 지방 세포6로분화한다. 최근 단세포 RNA-시퀀싱(scRNA-seq) 연구는 이러한 지방 선조 세포(APC) 집단(ASC 및 preadipocytes)이 상당한 분자 및 기능성 이질성7,8,9,10,11,12를나타낸다는것을 보여준다. 예를 들어, ASC는 감소된 사이포겐화 분화 능력을 표시하는 동시에, 또한 지방세포7에비해 더 높은 증식 및 확장 기능을 나타낸다. 추가 분자 다름은 ASC 및 preadipocyte 인구 내에서 보고됩니다, 그러나 이 다름의 기능적인 관련성은 불분명남아7. 함께, 이러한 데이터는 지방 선조 세포 풀의 복잡성을 강조하고 더 나은 이해하고 이러한 중요한 세포 인구를 조작하기 위해 도구를 개발하고 표준화 할 필요성을 강조한다.
이 프로토콜은 단일 세포 연구 (scRNA-시퀀싱) 및 세포 배양을 포함하여 민감한 다운스트림 분석에 적합한 마우스 상피 지방 패드에서 높은 생존성 Sca1+ 지방 전구 집단의 분리를 자세히 설명합니다. 상고체 지방 패드의 격리 및 해리는 이전에 설명된 바와 같이 수행되었다7,13 격리 된 APC의 생존가능성을 개선하는 약간의 수정. 간단히 말해서, 상고성 지방 패드에서 해리된 세포는 Sca1에 대한 항체, ASC 및 프리디포사이클6,7및기타 계보(Lin) 마커모두에 대한 마커로 염색됩니다: Ter119(적혈구), CD31(내피 세포), CD45(leukocytes). 실행 가능한 Sca1+/ter119-/CD31-/CD45-/DAPI- 세포는 형광 활성화 셀 정렬 (FACS)에 의해 정렬됩니다. 중요한 것은, 이 프로토콜은 ASC 및 프레디포세포7내의 기능적으로 이질적인 하위 집단을 확인한 최근 단세포 RNA 시퀀싱 연구에서 보고된 실행 가능한 Sca1 +/Lin-분면 전구 세포의 성공적인 격리 및 분석에 의해 검증되었다.

<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

당뇨병, 심장병 및 암과 같은 비만 및 대사 장애는 모두 극적인 지방 조직 리모델링과 관련이 있습니다. 조직 거주자 지방 선조 세포 (APC)는 조직 항상성에 중요한 역할을하고 조직 병리학에 기여할 수 있습니다. 단세포 RNA-염기서열 분석 및 단세포 프로테오믹스를 포함한 단일 세포 분석 기술의 사용이 증가하고 있으며, 인구 또는 조직 전반의 변화의 맥락 내에서 개별 세포 발현 변화의 전례 없는 해결을 허용함으로써 줄기/전구 세포 분야를 변화시키고 있다. 이 문서에서는 마우스 부피 지방 조직을 해부하고, 단일 지방 조직 유래 세포를 분리하고, 형광 활성화 세포 정렬(FACS)을 수행하여 실행 가능한 Sca1 +/CD31-/CD45-/Ter119- APC에 대한 상세한 프로토콜을 제공합니다. 이러한 프로토콜을 통해 조사관은 단일 세포 RNA 염기서열분석과 같은 다운스트림 분석에 적합한 고품질 APC를 준비할 수 있습니다.

4개월 된 남성 FVB 마우스는 이 실험에서 사용되었다. FSC/SSC 플롯을 사용하여 파편과 이중을 제외한 후 실행 가능한 세포(DAPI-인구)가 게이트되었고, 그 다음으로 APC+/FITC-인구(도1)의선택이 뒤따랐다. DAPI, APC 및 FITC 게이트는 스테인드 컨트롤을 기반으로 그려졌습니다. 게이팅 전략은 그림 1에표시됩니다.
1h ?...

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

우리는 형광 활성화 세포 분류를 사용하여 마우스 전도성 지방 패드에서 매우 실행 가능한 지방 전구 세포를 분리하는 간단한 방법을 제시한다.

마우스 전형 지방 조직에서 지방 전구의 준비

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

5.6K 조회수.  Mayo Clinic. 우리는 형광 활성화 세포 분류를 사용하여 마우스 전도성 지방 패드에서 매우 실행 가능한 지방 전구 세포를 분리하는 간단한 방법을 제시한다.

비디오: 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.

천장 문화 성숙한 지방 세포의 탈분화를 통해 인간 된 지방 줄기 세포의 생성

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

연구

발생학

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.

돼지 피하 지방이 조직에서 분리 및 지방이 유래 줄기 세포의 분화

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 ...

유전학

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.

절연, 확장, 및 Adipogenic CD34 + CD31 + 인간의 Omental 및 피하 지방 조직에서 내 피 세포의 유도

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

면역학 및 감염병학

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.

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.

신생아 마우스에서 기본 백색및 갈색 프레드포스세포의 격리 및 분화

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

생물학

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.

1차 배양에서 분화된 마우스 지방세포: 인슐린 저항성 모델

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

Murine Perivascular Adipose Tissue에서 분리된 지방세포(preadipocytes) 배양

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.

저자 스포트라이트: 마우스 혈관 주위 지방 조직에서 SVF 유래 지방세포의 분리, 배양 및 지방형성 유도의 중요성 

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.

Isolation, Culture, and Adipogenic Induction of Stromal Vascular Fraction-derived Preadipocytes from Mouse Periaortic Adipose Tissue(마우스 대동맥 주위 지방 조직에서 기질 혈관 분획 유래 지방세포의 분리, 배양 및 지방 유도)

마우스 전형 지방 조직에서 지방 전구의 준비

요약

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천장 문화 성숙한 지방 세포의 탈분화를 통해 인간 된 지방 줄기 세포의 생성

돼지 피하 지방이 조직에서 분리 및 지방이 유래 줄기 세포의 분화

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

절연, 확장, 및 Adipogenic CD34 + CD31 + 인간의 Omental 및 피하 지방 조직에서 내 피 세포의 유도

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

신생아 마우스에서 기본 백색및 갈색 프레드포스세포의 격리 및 분화

1차 배양에서 분화된 마우스 지방세포: 인슐린 저항성 모델

Isolation, Culture, and Adipogenic Induction of Stromal Vascular Fraction-derived Preadipocytes from Mouse Periaortic Adipose Tissue(마우스 대동맥 주위 지방 조직에서 기질 혈관 분획 유래 지방세포의 분리, 배양 및 지방 유도)

Isolation, Culture, and Adipogenic Induction of Stromal Vascular Fraction-derived Preadipocytes from Mouse Periaortic Adipose Tissue(마우스 대동맥 주위 지방 조직에서 기질 혈관 분획 유래 지방세포의 분리, 배양 및 지방 유도)

Isolation, Culture, and Adipogenic Induction of Stromal Vascular Fraction-derived Preadipocytes from Mouse Periaortic Adipose Tissue(마우스 대동맥 주위 지방 조직에서 기질 혈관 분획 유래 지방세포의 분리, 배양 및 지방 유도)