Name: induction and testing of hypoxia in cell culture
Uploaded: 2011-08-12T13:00:00
Duration: 7 min 1 s
Description: Watch this Scientific Journal Video about Induction and Testing of Hypoxia in Cell Culture at JoVE.com

1. CoCl이 솔루션 유도 Hypoxia 코발트 (II) 염화물 hexahydrate은 (CoCl 2 • 6H 2 O, MW = 237.9) hypoxia - inducible 인자 - 1.3 8 화학 유도합니다. 이 제품은 명확하고 붉은 솔루션을 항복, 물 (100 MG / ML)에 용해됩니다. <ol><li> 무균 DD 물에 25mM 재고 솔루션을 준비, (사용하기 전에 바로 준비) </li><li> hypoxia를 유발하여 일반 세포 배양 매체 100μM의 최종 농도에서 CoCl 2를 사용하십시오. </li><li> 당신의 세포에 CoCl이 포함된 미디어를 추가하고 기존의 인큐베이터에 24시간에 대한 문화를 품다 (37 ° C; 5% C0 2). </li></ol> 위의 농도는 우리가 테스트를했지만 각 세포주 마약 관련 독성을 제한하고 분석을 최적화하기 위해 다양한 배양 시간뿐만 아니라 다양한 농도 (복용 종속 곡선을 설정)에서 테스트해야하는 세포 라인에 적용됩니다. 2. 모듈형 보육 회의소에서 유도된 Hypoxia <ol><li> 적어도 두 개의 동일한 (트윈 문화) 세포의 문화를 준비합니다. 세포 배양 Flasks, 접시 또는 문화 요리하실 수 있습니다. 챔버를 열려면 먼저 챔버 (hypoxic 가스를 정화 주입 / 튜브 사용)하고 부드럽게 철강 링 클램프를 열고 연결된 튜브에있는 두 개의 하얀 플라스틱 클램프를 엽니다. </li><li> 클램프를 풀어. 뚜껑과 쟁반 이제 제거할 수 있습니다. </li><li> hypoxic 챔버에서 세포 배양을 놓습니다. 또한 문화의 적절한 가습을 제공하기 위해 챔버에 멸균 물이 들어있는 페트리 접시를 놓습니다. </li><li> 컨트롤로 normoxia에서 &quot;쌍둥이&quot;세포 배양를 놓습니다. </li><li> 귀하의 트레이가 안전하게 움직이지 않고 다음의 뚜껑이있는 곳에는 가까이 있는지 확인합니다. 제대로 챔버의 hermetical 폐쇄를 보장하고 닫습니다 철강 링 클램프 위치를. </li><li> hypoxia을 만들려면, 1 %의 O 2 기체 혼합물을 포함하는 &quot;hypoxia 탱크&quot;로 튜빙을 연결합니다. 당신의 탱크에 연결된 유량계가있다면 귀하의 챔버는 직접 (가스 탱크 - 유량계 - 챔버)에 연결됩니다. 우리는 감독 당국에 통합 유량계를 사용합니다. </li><li> 7-10분 분 동안 당 20 리터 유량의 가스 탱크를 열어 챔버를 플러시 이렇게하려면 챔버와 미디어의 모든 산소 존재가있다면 대부분을 제거하는 것이 중요하다, 그리고 신속하게 해제 가스 흐름과 완전히 흰색 클램프를 모두 닫아 챔버를 닫습니다. </li><li> 시간의 원하는 기간 동안 기존의 인큐베이터로 챔버를 반환합니다. 2 시간 후 물을 반복 - 당신이 큰 문화를 사용하는 경우, 1 드 가스 문화 미디어가 수 있습니다. 위의 단계는 생성자 추천 &#39;(빌럽스 - 로텐버그, 부동산)을 기반으로합니다. 당신은 가스 탱크가 제대로 모든 시간에 안전하게 있는지 확인합니다. </li></ol> 참고 : <ul><li> 그것이 1-3시간 후 챔버 번 다시 가스 좋습니다 미디어에 포함된 O 2를 제거하기 위해서는. </li><li> 이상 48시간보다 배양 시간에 그것은 또한 다시 가스 챔버에 좋습니다. </li><li> O 2의 측정 셀 / 챔버에 포함된 수 있도록 산소 센서 (전극 / 혈압계) 챔버 / 세포 O 2가 포함된)의보다 정확한 측정을 제공합니다. </li><li> 시간 곡선을 만들어 여러 시간 길이에 Culturing 세포는 특정 세포에서 HIF - 1α의 최적의 시간 표현을 결정하는 데 도움 것입니다. </li></ul> 3. Hypoxia 평가 <ol><li> 서양 얼룩에 의해 HIF - 1 α 검출. <ol><li> 다시 열고 챔버는 섹션 2.2에서 설명한 즉시 얼음에 문화를 놓으십시오. </li><li> 접시에 5% SDS 솔루션 hypoxia 처리 및 비 치료 세포를 Lyse하면 다음 튜브에 sonicate, spindown 세포 파편을 세포 lyzate를 전송하고 표면에 뜨는를 수집합니다. </li><li> BCA 키트를 사용하여 측정 단백질 농도는 5 분 95시 로딩 버퍼와 난방 ° C를 추가하여 샘플을 준비합니다. </li><li> 8% SDS - polyacrylamide 젤 및 Nitrocellulose의 점막에 전송에 Electrophorese 단백질. </li><li> 1 시간이나 쉐이크 4 ° C에서 하룻밤 상온에서 5% 아닌 지방 건조 우유를 포함하는 PBS에 막과 0.1 % 트윈 20 차단합니다. </li><li> 4 같은 버퍼에 방지 HIF - 1α 기본 단클론 항체 (1 / 600)와 함께 밤새 Immunoblot ° C. </li><li> PBS는 PBS에서 HRP - 이차 항체 (1 / 2000) + 0.1 % 45 분 트윈 20과 실내 온도, 5 분마다 시간과 부화에 트윈 20 0.1 %를 포함하는 3 번 씻으십시오. </li><li> PBS는 실온, 5 분 / 시간에 트윈 20 0.1 %를 포함하는 3 번 씻으십시오. </li><li> 단백질 밴드를 표시 피어스 ECL Kit 및 X - 선 필름을 사용 </li></ol></li><li> Hypoxia 시브 요소 (HRE). HIF - 1α는 Hypoxia 레굴라팅 요소 (HRE)라는 순서에 바인딩하여 수많은 유전자 (즉 VEGF, EPO)을 규정합니다. 마커 유전자에 관련된 HRE를 사용하여 It 9 HIF 활동을 감지할 수 있습니다. 이 방법을 사용하려면 먼저 귀하의 세포에 적응 transfection 방법을 사용하여 HRE - 루시페라제 플라스미드와 세포 transfect해야합니다. 예를 들어, 우리는 Lipofectamine 2000를 사용합니다. 세포는 hypoxia과 normoxia에 incubated 수 전에 최소한 4 시간 transfected해야합니다. 이번이 첫 번째 단계는 hypoxia에 의해 영향을받지 않는다 있도록 DNA가 세포를 입력할 수 있습니다. 루시페라제 신호 루시페라제 어세이 키트를 사용하여 감지됩니다. <ol><li> 모듈 챔버에서 hypoxia에 HRE - transfected 세포를 품어 또는 CoCl 2 incubated HRE - transfected 세포를 사용 normoxia 컨트롤을 포함합니다. </li><li> 원하는 잠복기 후, 세포의 성장 매체를 제거합니다. </li><li> PBS로 세포를 린스, PBS를 제거합니다. </li><li> 문화 접시에 400μl 1X 용해 시약을 조제하고 첨부된 세포를 긁어 다음, 마이크로 튜브로 전송할 수 있습니다. </li><li> 간단한 원심 분리하여 펠렛의 잔해. </li><li> 세포의 믹스 20μl는 루시페라제 분석 시약의 100μl로 뜨는 lysates과 luminometer를 사용하여 발광을 측정합니다. </li></ol></li></ol> 4. 대표 결과 : K562 세포 (인간 백혈병 세포주)에서 normoxia에 비해 낮은 산소 교양 이일, hypoxia 서양 얼룩 (그림 1)에 의해 감지되었습니다 HIF - 1α 단백질의 증가를 유도. HRE - 루시페라제은 hypoxia에 교양 (배아 신장 세포 라인) 293 세포를 수정이 중요한이 HIF - 1α 활동의 증가 hypoxic 세포에서 발견되었다 (그림 2). 사용 <img src="/files/ftp_upload/2899/2899fig1.jpg" alt="그림 1"/> 그림 1 : hypoxic 세포에서 HIF - 1α 늘립니다. K562 세포는 48 시간을위한 normoxia 및 hypoxia (hypoxic 챔버)에 문화되었으며 HIF - 1α에 대한 특정 항체를 사용하여 서양 얼룩으로 분석했다. 굴지에 대한 항체 특정은 로딩 제어 사용되었다. <img src="/files/ftp_upload/2899/2899fig2.jpg" alt="그림 2"/> Figure2 : HIF - 1α 활동. HRE - 루시페라제는 293 세포는 48 시간을위한 hypoxia과 normoxia에 교양되었습니다 수정한 다음 루시페라제 분석 키트와 luminometer를 사용하여 루시페라제 신호를 감지하는 lysed. 결과는 상대적인 발광 장치 (RLU)로 표현됩니다.

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hypoxic 조건에서 세포 증식과 생존은 세포 종류에 따라 많이 다릅니다. 따라서, 당신은 전화 번호 또는 귀하의 실험에 대한 충분한 세포 / 단백질을해야합니다 있는지 확인으로 시작 문화 접시의 수를 조정해야합니다. 코발트 클로라이드 방법은 저렴한 가격과 빠른 될 수있는 장점이 있습니다. 이 제품은 HIF-1/3α 유도하여 hypoxia 일일뿐만 아니라이 제품은 그것의 &#39;모방 - 독립적으로 특정 세포의 기능과 표현형에 미칠 수있는 다른 어떤 효과를 선택하여 프로젝트에 맞게되어 있는지 확인, 다른 유전자를 조절 할 수 hypoxic &quot;효과. 또한 hypoxia을 모방하는 데 사용되는 다른 약물은 Deferoxamine 메실레이트 (돌연사 했을까, 100 μm의 최종 농도에서 사용)입니다. 이러한 약물의 사용은 experimentator가 &quot;hypoxic 상태&quot;에 영향을주지 않고 문화 판 / 요리 / 술병을 여러 번 열 수 있습니다. 가스 혼합물에 포함된 산소의 수준은 hypoxia 값이 조직 및 세포 유형에 따라 다릅니다로 실험 및 세포 종류에 따라 달라질 수 있습니다. 사실, 몇몇 세포는 O 2 기타 필요한 1 % 미만 O 2 hypoxic로 5 % hypoxic 있습니다. 5 % CO 2는 문화의 산도를 안정화하기 위해 가스 혼합물에 추가, 가스의 나머지 부분은 보통 질소이다. Hypoxic 챔버 스는 독립적으로 산소 장력의 세포 행동을 대체 할 수 의약품을 사용하지 않는 장점이 있습니다. 산소가 각 오프닝에서 챔버를 다시 입력하고 따라서 hypoxia을 lessens 그러나, 실험의 모든 유형의 작업을 수행할 수 있습니다. 귀하의 실험이 요소를 고려해야한다 hypoxia / 다시 산소 일부 세포 유형에 영향을 미칠 수있는 특정 조건입니다. 대안 hypoxia 워크 스테이션 (미리 혼합 가스 탱크 또는 가스 혼합 시스템에 연결된) 또는 실험 미디어를 변경하고 지속적인 hypoxic 환경에서 세포를 조작할 수 있도록 큰 hypoxia 인큐베이터 (글러브 박스와 hypoxia 처리 챔버)를 사용하는 것입니다. 다양한 hypoxic 챔버 스는 지난 10 년간 상용화되었습니다 귀하의 연구실에 사용, 프로젝트, 공간, 크기 및 예산에 따라 선택되어야합니다. 항상 올바른 문화 조건을 보장하여 챔버 잘 사용하고 조건을 보장합니다. 일반적으로 챔버를 사용하는 경우, hypoxia의 수준은 다양한 가스 혼합 (예 : 1퍼센트, 5 % 10 % 산소)를 선택하여 변조된 수 있습니다. HIF - 1α의 검색에 대해서는 아는 것이 중요합니다 일부 암 세포주 normoxia에서 HIF - 1α 표현. 그러므로 이러한 세포 라인에서 HIF의 기초 수준을 결정하는 normoxia - 컨트롤을 사용하는 것이 중요합니다. HIF - 1α는 또한 세포 자극이나 스트레스 다음 이외의 악성 세포 normoxia에 존재하실 수 있습니다. 이것은 또한이 세포는 굶어 죽었을 경우 발생하는 휴대폰의 문화가 적절하게 공급, 유지되어 있는지 확인 수 있습니다.

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th></tr><tr><td> 안티 HIF - 1α </td><td> Invitrogen </td><td> 458400 </td></tr><tr><td> 코발트 (II) 염화물 </td><td> 시그마 </td><td> C8661 - 25G </td></tr><tr><td> 보육 상공 회의소 </td><td> 빌럽스 - 로텐버그 </td><td> MIC - 101 </td></tr><tr><td> HRE - 루시페라제 플라스미드 </td><td> Adgene </td><td> 26,731 플라스미드 </td></tr></tbody></table>

induction and testing of hypoxia in cell culture

Hypoxia는 기관, 조직 또는 세포에 산소의 감소 또는 결여로 정의됩니다. 산소 장력의이 감소는 산소의 감소 공급 장치 (원인이 부족한 혈관 네트워크 결함이 혈관과 빈혈을 포함)하거나 공급 장치 (갑자기 높은 세포 증식 률에 의한)에 상대적으로 산소의 증가 소비로 인해 수 . Hypoxia는 1-3, 류마티스 관절염, 죽상 경화증 등 고체 암에서와 같이 physiologic 또는 pathologic 수 있습니다 ... 각 조직과 세포는이 새로운 조건에 적응하기 위해 다른 기능이 있습니다. hypoxia 동안, hypoxia inducible 요인 알파 (HIF)를 안정화와 같은 산소 4 angiogenesis 또는 운송에 관련된 사람 등 다양한 유전자를 조절합니다. 이 단백질의 안정화 따라서 HIF가 일상적으로 hypoxia 5-7위한 화면으로 사용됩니다 감지, hypoxia의 특징이다. 이 문서에서는, 우리는 이들 세포의 hypoxic 상태를 평가하는 포유 동물 세포 배양 및 간단한 테스트에 hypoxia를 유도하기 위해 두 가지 간단한 방법을 제안합니다.

Watch this Scientific Journal Video about Induction and Testing of Hypoxia in Cell Culture at JoVE.com

Induction and Testing of Hypoxia in Cell Culture

여기서 우리는 문화의 hypoxic 상태를 평가하는 세포 문화와 간단한 테스트에 hypoxia를 유발하는 간단한 방법을 제안합니다.

세포 배양의 유도와 Hypoxia의 시험

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

induction-and-testing-of-hypoxia-in-cell-culture

Research

JoVE Journal

Biology

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

마우스 신경 줄기 세포 엽 성의 전구 물질의 문화

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

연구

생물학

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin. Such reactions can also be induced against autoantigen, for example to myelin basic protein (MBP) in rats with experimental autoimmune encephalomyelitis induced with MBP, an animal model for multiple sclerosis (1).
Here we demonstrate how to induce an adoptive DTH reaction in Lewis rats. We will first stimulate ovalbumin-specific T cells in vitro and inject these activated cells intraperitoneally to naive rats. After allowing the cells to equilibrate in vivo for 2 days, we will challenge the rats with ovalbumin in the pinna of one ear, while the other ear wil receive saline. The inflammatory reaction will be visible 3-72 hours later and ear thickness will be measured as an indication of DTH severity.

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.

고양이의 입양 지연 유형 과민성의 유도 및 모니터링

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

작은 분자와 유도 Pluripotent 인간 배아 줄기 세포로부터 인간 심장 엽 성의 전구 물질과 Cardiomyocytes의 효율 유도

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

쥐 간장 세포의 분리 및 차 문화

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

이식편 문화 치아 세균의 개발을 다음과 같은 사항에 대해 슬라이스 문화 방법

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

근육 위성 세포의 분리, 문화, 및 이식

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

저산소증에 따라 단일 세포 수준에서 글로벌 RNA의 합성 분석

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

은 C-FOS 단백질 면역 조직 검색 : 특정 생리적 반응에 관여 중앙 진학의 마커로서 유용한 도구<em&gt; 생체</em&gt; 및<em&gt; 전의 VIVO</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

RNA 결합 단백질의 검출에 의해<em&gt; 체외</em지방 세포 문화&gt; RNA 풀다운

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.