이 연구는 국립 심장, 폐, 혈액 연구소 그랜트 HL- 103542, 그리고 CAP에 BCH 마취 연구 재단의 고유 선구자 상에 의해 지원되었다.

준비 <ol><li> 한 M K-HEPES 원액을 (KOH와 7.2 산도를 조정) 준비합니다. </li><li> 0.5 M K-EGTA 주식 솔루션 (KOH 8.0의 산도를 조정) 준비합니다. </li><li> 한 M KH 2 PO 4 원액을 준비합니다. </li><li> 한 M MgCl2를 원액을 준비합니다. </li><li> 균질화 완충액 (pH 7.2) 300 MM의 크로스, 10 mM의 K-HEPES 및 1 ㎜ K-EGTA를 준비합니다. 4 ℃에서 보관하십시오. </li><li> 호흡 버퍼 250 MM의 자당을 준비, 2 mM의 KH 2 PO 4, 10 mM의 MgCl2를, 20 mM의 K-HEPES 완충액 (pH 7.2) 및 0.5 mM의 K-EGTA (산도 8.0). 4 ℃에서 보관하십시오. </li><li> 의 NaCl 80g,의 KCl 2g, 나 2 HPO 4의 14.4 g, 1 L 더블 증류수 H 2 O (산도 7.4)에 KH 2 PO 4 2.4 g을 용해시켜 10 배 PBS 원액을 준비합니다. </li><li> 1 L 더블 증류수 H 2 O로 100 ML의 10 배 PBS 피펫 팅에 의해 1X PBS를 준비 </li><li> 서브 틸리의 4 밀리그램을 무게에 의해 서브 틸리 스톡 준비1.5 ML의 microfuge 튜브. 사용할 때까지 -20 ° C에서 보관하십시오. </li><li> 1.5 ml의 미세 원심 분리 튜브에 BSA의 20 밀리그램을 계량하여 BSA 콘텐츠를 준비합니다. 사용할 때까지 -20 ° C에서 보관하십시오. </li></ol> 미토콘드리아 절연 (그림 1) <ol><li> 분리 직전에, 균질화 완충액 1 ㎖에 용해 서브 틸리. </li><li> 분리 직전에, 균질화 완충액 1 ml의 BSA를 녹인다. </li><li> 얼음에 50 ML 원뿔 원심 분리 관에 1X PBS에서 6mm 생검 샘플 펀치와 저장소를 사용하여 두 개의 신선한 조직 샘플을 수집합니다. </li><li> 버퍼 균질화 빙냉 5 ㎖를 함유 해리 C 관 조직이 6mm 펀치를 전송합니다. </li><li> 티슈 dissociator에 해리 C 튜브 피팅에 의해 조직을 균질화하고 미리 설정된 미토콘드리아 분리 사이클 (60 초간 균질화)을 선택한다. </li><li> 얼음 양동이에 해리 C 튜브를 제거합니다. </li><li> 에 서브 틸리 원액의 250 μl를 추가파쇄는 반전하여 혼합하고 10 분간 얼음에 균질 배양. </li><li> 균질화 버퍼 얼음과 사전에 젖은 필터에 50 ML 원뿔 원심 분리 관에 40 μm의 메쉬 필터를 넣고 얼음에 50 ML 원뿔 원심 분리 관에 균질를 필터링 할 수 있습니다. </li><li> 여액에 새로 제조 된 BSA 원액의 250 μl를 추가하고 반전으로 섞는다. 참고 : 미토콘드리아 단백질 결정이 필요한 경우이 단계를 생략하십시오. </li><li> 균질화 버퍼 얼음과 사전에 젖은 필터에 50 ML 원뿔 원심 분리 관에 40 μm의 필터를 넣고 얼음에 50 ML 원뿔 원심 분리 관에 균질를 필터링 할 수 있습니다. </li><li> 균질화 버퍼 얼음과 사전에 젖은 필터에 50 ML 원뿔 원심 분리 관에 10 μm의 필터를 넣고 얼음에 50 ML 원뿔 원심 분리 관에 균질를 필터링 할 수 있습니다. </li><li> 10 분에서 9,000 XG에서이 미리 냉장 1.5 ML의 microfuge 튜브와 원심 분리기 여과 액을 전송4 ºC. </li><li> 상층 액을 제거하고 다시 중단하고 차가운 얼음 호흡 버퍼의 1 ml의 알약을 결합한다. </li></ol> ATP 분석 주 : ATP 발광 분석법은 ATP 분석 키트를 사용하여 수행 될 수있다 절연 미토콘드리아 대사 활성을 결정한다. 프로토콜은, 시약 및 표준 분석 키트에 공급되었다. 절차의 개요에 대하여 설명한다. <ol><li> RT에 키트 시약을 평형. </li><li> 이중 증류수 1,170 μL에서 동결 건조 된 ATP 펠렛을 용해시켜 10 mM의 ATP 원액을 준비합니다. ATP에게 얼음에 표준 원액 및 준비 미토콘드리아 샘플을 저장합니다. </li><li> 동결 건조 된 기질 용액 바이알에 기질 완충액 5 mL를 추가한다. 부드럽게 믹스와 어둠 속에서 배치합니다. </li><li> 블랙, 불투명 하단, 96 웰 플레이트의 모든 우물에 호흡 버퍼의 100 μl를 추가합니다. </li><li> 각 준비된 샘플에서 잘 O를 미토콘드리아의 10 μl를 추가FA 블랙, 불투명 하단, 96 웰 플레이트. 참고 : 시료는 세중 도금된다. 표준 및 음성 대조군 (호흡 버퍼)에 대한 세 우물에 대한 행을 포함합니다. </li><li> 표준 및 컨트롤을 포함한 모든 웰에 포유 동물의 세포 용해 용액 50 μl를 추가합니다. </li><li> 125 rpm에서 진탕에 5 분 동안 37 ºC에서 접시를 품어. </li><li> 배양 동안 10 mM의 ATP 원액 0.1 밀리미터, 0.05 밀리미터, 0.01 밀리미터, 0.005 밀리미터, 0.001 밀리미터, 및 0.0001 mM의 ATP의 농도 ATP 표준을 준비합니다. 얼음에 보관 기준. </li><li> 배양 후, 플레이트지도에 표시된 우물을 대응하는 ATP 기준의 10 μl를 추가합니다. 참고 : 표준은 중복 수행됩니다. </li><li> 각 웰에 재구성 된 기질 용액 50 μl를 추가합니다. </li><li> 125 rpm에서 5 분 동안 진탕 기에서 37 ºC에서 접시를 품어. </li><li> 분광 광도계로 연결된 컴퓨터에서 열기 GEN5 1.11 소프트웨어입니다. </li><li> &quot;에서새 항목을 만들 실험 &quot;&quot;클릭 &quot;. </li><li> &quot;기본 프로토콜&quot;을 클릭합니다. &quot;확인&quot;을 클릭합니다. </li><li> 왼쪽 열에서 다음, &quot;절차&quot;를 &quot;프로토콜&quot;을 선택합니다. </li><li> &quot;지연&quot;을 선택합니다. 0시 10분 0초로 설정합니다. &quot;확인&quot;을 클릭합니다. </li><li> 선택 &quot;읽기&quot;. 드롭 다운 메뉴에서 &quot;발광&quot;를 선택합니다. 다음에 다른 설정을 조정 : 유형 엔드 포인트, 통합 시간 공을 읽는다 01.00 MM : SS.ss, 필터 1 세트, 배출 홀, 광학 위치 최고, 감도 100, 최고 프로브 수직 1.00 mm 오프셋. </li><li> 판을 분석 할 준비가되면, 맨 윗줄에있는 &quot;판을 읽고&quot;아이콘을 클릭합니다. &quot;읽기&quot;를 클릭합니다. 분광 광도계가 열리면 왼쪽 상단 모서리에 잘 A1 트레이에 접시를 놓습니다. 온도에 상자가 열립니다. &quot;읽기&quot;를 클릭합니다. 참고 : 값이 높을수록 증가 ATP 수준 높은 신진 대사 활동과 상관 관계. </li></ol>

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모든 동물은 현재 기관 가이드 라인에 따라 처리 하였다. 우리는 공개 경쟁 금융 이익을 더이 없습니다.

성공적으로 미토콘드리아 생존을 유지하기 위해 얼음에 모든 솔루션과 조직 샘플을 유지하는 것이 필수적입니다이 프로토콜을 사용하여 미토콘드리아를 분리합니다. 얼음에서 유지하더라도, 절연 미토콘드리아는 19 시간에 걸쳐 기능적 활성의 감소를 나타내는 것이다. 우리는 솔루션 및 추가가 미리 준비하는 것이 좋습니다. 우리는 - 무게 사전 및 1.5 ml의 미세 원심 분리 튜브에 4 밀리?...

A correction was made to: <a href="http://www.jove.com/video/51682">Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration.</a> An optional step was added to step 7 of the &#39;Mitochondrial Isolation&#39; section of the protocol. The step is:
1. Optional in the case that the tissue is fibrous: Centrifuge the solution at 750 x g for 4 min.

A correction was made to: Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration.

Erratum: Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

미토콘드리아는 적혈구를 제외한 체내 모든 세포에 존재하는 세포 및 중요한 대사 과정 1-4의 다수에 관여한다. 때문에 이러한 많은 기능의 미토콘드리아 손상이 해로운 영향 삼을 수 있습니다. 미토콘드리아 기능 부전 여러 미토콘드리아 분리 방법을 조사하기 위해서 설명되었다. 1940 5-8로 미토콘드리아 격리 날짜의 최초의 공표를 차지한다. 제 문서화 시도가 저속 5,8에서 염 용액에 원심 분리 박격포 간 조직을 분쇄하여 미토콘드리아 아이솔레이션을 보여 주었다. 나중에, 다른 그룹은 원래의 절차에 따라 팽창 차등 원심 6-8에 기초하여 조직 분획을 보여 주었다. 이러한 초기 방법은 종종 균질화, 및 / 또는 차등 원심 9-15 통합 현재 기술의 기초를 형성. homogenizati의 수단계와 원심 분리 프로토콜마다 다릅니다. 이러한 반복적 인 단계들은 미토콘드리아 분리를 위해 시간을 증가시키고, 궁극적으로 생존 능력을 감소시킨다. 제대로 10,16 제어하지 않을 경우 또한, 수동 균질화는 미토콘드리아 손상과 일치하지 않는 결과가 발생할 수 있습니다. 최근에, 우리는 심근 조직 (17, 18)에 이식 미토콘드리아를 분리 균질화 및 차등 원심 분리를 사용했다. 이 긴 분리 절차를 수행하는 데 약 90 분을 요구하고이 방법의 임상 적용 가능성 때문에 제한되었다. 임상 및 수술 적 치료에서의 급성 치료 적 사용을 허용하기 위해, 우리는 30 분 미만으로 수행 될 수있는 신속한 미토콘드리아 분리 과정을 개발 하였다. 이 프로토콜의 주요 장점은 표준화 된 조직 해리가 균일하고 쉽게 수동 균질화 달성되지 않은 조직의 일관성 균질화 수 있다는 것입니다. 와 T에서이온 차등 원심 분리 대신에 차동 여과의 사용은 시간이 많이 소요되고 고도로 정제 실용적이고 유능한 미토콘드리아 호흡의 더 신속한 분리를 허용 반복 원심 분리 단계를 제거한다. 미만 30 분에 실행 가능하고 호흡 능력 미토콘드리아를 분리 할 수​​있는 능력은 임상 적 적용이 가능합니다. 이 분리 프로토콜은 관상 동맥 우회 이식 수술 (CABG) 등의 치료 과정에 사용 목적이 가능성이있다.

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			<td>267110</td>
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rapid isolation and purification of mitochondria for transplantation by tissue dissociation and differential filtration

차동 원심 분리 및 / 또는 피콜 구배 원심 분리를 사용하여 이전에 기술 된 미토콘드리아 분리 방법은 완료 60-100 분을 필요로한다. 우리는 상업적 조직 dissociator 및 차등 여과하여 포유류의 조직 검사에서 미토콘드리아의 신속한 분리를위한 방법을 설명한다. 이 프로토콜에서 수동 균질화는 조직 dissociator의 표준화 균질화 사이클로 대체됩니다. 이 균일하고 쉽게 수동 균질화 달성되지 않은 조직의 일관성 균질화 수 있습니다. 티슈 해리 다음, 균질 반복 원심 분리 단계를 제거 나일론 메쉬 필터를 통해 여과된다. 그 결과, 미토콘드리아 절연은 30 분 미만으로 수행 될 수있다. 이 분리 프로토콜은 0.18 ± 0.04 g (습 중량) 조직 샘플에서 약 2 × 10 10 실행 가능하고 호흡 능력 미토콘드리아를 얻을 수 있습니다.

티슈 해리 및 차등 여과를 이용하여 미토콘드리아 격리에서 절차 단계를 개략적 인 그림이도 1에 도시된다. 전체 절차 시간을 30 분 미만이다. 조직 샘플은 6mm 생검 펀치를 사용하여 수득 하였다. 조직의 무게는 0.18 ± 0.04 g (습 중량)이었다. 입경 계수에 의해 결정으로서 단리 미토콘드리아의 수는 2.4이었다 × 10 10 ± 0.1 × 10 10 골격근 미토콘드리아...

Watch this Scientific Journal Video about Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration at JoVE.com

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

포유 동물 조직 생검에서 미토콘드리아의 신속한 분리를위한 방법을 설명한다. 쥐 간 또는 골격근 제제 상용 조직 dissociator 균질화하고, 미토콘드리아는 나일론 메쉬 필터를 통한 여과에 의해 미분을 분리 하였다. 대안적인 방법을 사용하여 100 분간 - 미토콘드리아 분리 시간은 60 비교 &lt;30 분이다.

조직 해리 차동 여과 이식 미토콘드리아의 신속한 분리 및 정제

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

rapid-isolation-purification-mitochondria-for-transplantation-tissue

Research

JoVE Journal

Biology

37.7K 조회수.  Beth Israel Deaconess Medical Center and Harvard Medical School.  포유 동물 조직 생검에서 미토콘드리아의 신속한 분리를위한 방법을 설명한다. 쥐 간 또는 골격근 제제 상용 조직 dissociator 균질화하고, 미토콘드리아는 나일론 메쉬 필터를 통한 여과에 의해 미분을 분리 하였다. 대안적인 방법을 사용하여 100 분간 - 미토콘드리아 분리 시간은 60 비교 

비디오: Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

분리 및 조혈 줄기 세포의 이식 (HSCs)

연구

생물학

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

유기 용매 및 초산없이 쿠매시 G - 250로 젤의 단백질 염색법

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

효모 세포 Mitochondria의 정화

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

의 분리 및 정제 Drosophila</em마그네틱 비드별로 정렬> 주변 뉴런

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA 정제에 의한 염색질 절연 (처프)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

Planarian의 부동화, 부분 조사 및 조직 이식

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

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 ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

분리 및 배양 세포 및 마우스 조직에서 미토콘드리아의 기능적 분석

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

핵산의 여과 분리 : 간단하고 신속한 DNA 추출 방법

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.