일이 I.K.H.P. 국가 건강 및 의료 연구 위원회 (GNT1125033 및 GNT1140187) 및 호주 연구 협의회 (DP170103790)에서 교부 금에 의해 지원 되었다

1입니다. Apoptosis 유도
<ol><li>5 분 및 모든 기존의 세포 파편을 제거 하려면 삭제 상쾌한 300 x g에서 원심 분리기 셀 샘플. 참고: 부착 셀을 사용 하는 경우 사전에 셀 씨 고 apoptosis 유도 전에 인산 염 버퍼 솔루션 (PBS) x 1 씻어.</li>
	<li>휴대폰 번호를 확인 하 고 세포를 수집 합니다. 참고: 분석 결과 후 절연에 따라 좋습니다 적어도 1 x 10의 시작 셀 번호7 셀을.</li>
	<li>1 x 106 셀/mL의 최종 농도 대 한 완전 한 미디어 (10% (vol/vol) 태아 송아지 혈 청, 50 IU/mL 페니실린, 50 µ g/mL 스 혼합물을 포함 하는 각 매체)에서 resuspend.</li>
	<li>Aliquot ~ 106 셀 6 잘 플레이트의 당 x 2.</li>
	<li>Apoptosis를 유도 하는 접시 뚜껑을 제거 하 고 150 mJ/cm2 UV irradiator를 사용 하 여 셀을 비추는. 이 약 30-60 s를 취해야 한다. 참고: 이전에 방사선,6 셀 ~0.5 x 10 'Untreated' 셀 컨트롤에 대 한 유지 됩니다 확인 합니다. 참고: Apoptosis 또한 안티 Fas 또는 혈 청 기아6등 다른 방법을 통해 유도 될 수 있다.</li>
	<li>37 ° C, 5% CO2 셀 라인에 따라 2-8 시간 동안에 품 어.</li>
	<li>벤치 가기 가벼운 현미경을 사용 하 여, 시각화 apoptotic 형태학, blebbing, apoptopodia 형성, ApoBD 형성 등의 존재를 확인 하는 세포. 참고: 40 X 확대는 충분 한</li>
	<li>P1000 피 펫을 사용 하 여, 플라스틱 및 apoptotic 샘플을 수집 합니다.</li>
	<li>1 x PBS 가진 접시를 세척 하 고 나머지 샘플 최대 수확량을 위해 결합.</li>
	<li>수집 ~1/10th '전체 Apoptotic 샘플' (WAS)를 제어 합니다.</li>
	<li>'치료' 샘플을 수집 합니다.</li>
	<li>3000 x g 6 분에 WAS 및 Untreated 샘플 원심</li>
	<li>1 x PBS의 1 mL에 resuspend 하 고 얼음에 따로.</li>
	<li>ApoBD 절연, 어느 2 또는 3 단계 나머지 apoptotic 샘플 계속.</li>
</ol>2. ApoBD FACS 통해 격리
<ol><li>3000 x g 6 분에 전체 샘플 원심</li>
	<li>펠 릿을 중단 하지 않고는 상쾌한의 대부분을 제거 합니다.</li>
	<li>1 x A5 바인딩 버퍼, A5 FITC의 75 µ L 당 1 x 107 셀에-프로-3의 2 µ L의 1 mL를 포함 하는 얼룩 솔루션에서 resuspend.</li>
	<li>10 분 동안 실내 온도에 어둠 속에서 샘플을 품 어.</li>
	<li>1 x A5 바인딩 버퍼의 1-2 mL을 추가 하 고 원심 3000 x g 6 분 초과 얼룩 제거에서 샘플. 참고: 혼합된 셀 인구 또는 조직 샘플에 대 한 수행 단계 1 x A5 바인딩 버퍼에 셀 유형 전용 표식의 조합을 사용 하 여 얼룩이 지는 항 체와 3000 x g 6에서 원심 분리 하기 전에 (또는 제조 업체의 프로토콜에 의하여) 20 분 동안 얼음에 품 어 분입니다.</li>
	<li>FACS 버퍼의 3 ml에서 샘플 펠 릿 resuspend (1x PBS, 1 x A5 바인딩 버퍼, 10 %FSC, 2 mM EDTA) 1 x 107 셀 당.</li>
	<li>라운드-하단, 폴 리 프로필 렌 (cytometry) 튜브로 70 µ m 셀 스 트레이너를 통해 필터 및 샘플 얼음에, 어둠 속에서 계속.</li>
	<li>FACS 기계와 100 µ m 노즐을 사용 하 여 표준 설치를 수행, 드롭 지연 수행을 켜고 안정적인 흐름을 보장 합니다.</li>
	<li>샘플을 로드 하 고 ~ 1000 이벤트/s 수집 속도 설정 합니다.</li>
	<li>FSC, SSC, APC (TO-프로-3) 조정 하 고 (A5) FITC 전압 및 인구를 보장 하기 위해 FACS 플롯 내의 위치 이벤트 명확 하 게 분리 될 수 있다.</li>
	<li>20000 이벤트를 기록 합니다.</li>
	<li>제 4 부로 제어 전략을 설정 합니다.</li>
	<li>정렬 레이아웃에서 원하는 정렬 인구로 최종 ApoBD 게이트를 추가.</li>
	<li>샘플 수집 시작 되 고 ~ 250 µ L FACS 버퍼를 포함 하는 새로운 관으로 5000-10000 ApoBDs를 수집 하 여 테스트 정렬을 수행.</li>
	<li>뒤 시스템을 수행 하 고 정렬된 ApoBDs을 로드 합니다.</li>
	<li>취득 및 테스트-정렬 ApoBDs을 기록.</li>
	<li>ApoBD 순도 FSC (y 축) 대 a 5를 비교 하 여 ~ 99% 인지 확인 (x 축 이벤트). 참고: 다시 레이저 표백으로 인해 샘플을 분석 하는 경우에 약간 줄일 수 있습니다 A5 얼룩.</li>
	<li>고 순도 달성 되 면 원래 샘플을 로드 하 고 정렬 ApoBDs의 원하는 숫자를 얻을 때까지 계속. 참고: 필요한 경우 듀얼 정렬을 수행할 수 있습니다 동시에 ApoCells 및 ApoBDs를 분리. 참고: 시간의 긴 기간 동안 정렬할 때 좋습니다 잠복기 4 ° c.에 컬렉션 튜브</li>
	<li>정렬이 완료 되 면 정렬 후 ApoBDs의 작은 부분을 수집, ApoCells, Untreated, 및 WAS apoptosis의 유효성을 검사 하 여 정렬 후 순도 확인 후 정렬. 참고: 테스트 정렬 및 정렬 후 순도 크게 차이가 안 한다, 비록이 스트림 설정 및 안정성에 기반입니다.</li>
</ol>3. ApoBD 절연 차등 원심 분리를 통해
<ol><li>10 분에 대 한 300 x g에서 나머지 apoptotic 샘플 원심</li>
	<li>~ 500 µ L 셀 펠 렛을 방해 하지 않으려면 떠나 상쾌한 수집 하 고 새로운 15 mL 원뿔 튜브로 추가.</li>
	<li>나머지 500 µ L을 제거 하 고 (이 'ApoCell 농축 비율'을 나타냅니다 1 x PBS의 2 mL에 셀 펠 릿 resuspend.</li>
	<li>3000 g에서 20 분에 대 한 수집된 상쾌한 원심</li>
	<li>펠 릿에 대 한 확인 하 고 조심 스럽게 제거 상쾌한 (는 상쾌한 microvesicles 및 exosomes를 포함 하 여 작은 세포 외 소포를 포함할 수 있습니다).</li>
	<li>1 x PBS (이 대표는 ' ApoBD-농축 ' 분수)의 1 mL에 펠 릿 resuspend</li>
	<li>각 실용적, WAS, ApoCell-농축, 및 새로운 라운드-하단, 폴리스 티 렌 (cytometry) 튜브 샘플 ApoBD 농축의 100 µ L를 수집 합니다.</li>
	<li>2 x A5 바인딩 버퍼, 1: 100 A5-FITC, 및 1:1,000에-프로-3를 포함 하는 얼룩의 100 µ L를 추가</li>
	<li>어둠 속에서 10 분 동안 실내 온도에 품 어.</li>
	<li>Cytometry, apoptosis의 성공적인 유도 및 ApoBD 농축 분수의 순도 확인 하기 위해 위에서 설명한 제어 전략을 사용 하 여 샘플을 분석 합니다.</li>
</ol>4. Cytometry 전략 게이팅
<ol><li>플롯을-프로-3 (y 축) FSC (x 축) 모든 다른 비-permeabilized 이벤트 (TO-프로-3낮은/중간)에서 괴 사 성 세포 (TO-프로-3높은) 분리에 대 한.</li>
	<li>모든 비 permeabilized 이벤트를 선택 하 고 a 5에 대 한 SSC 플롯. 두 개의 인구 인구 1 (P1), SSC중간/높은,낮은/중간 셀 A5 및 인구 2 (P2), 다른 모든 이벤트를 포함 하 여 게이트.</li>
	<li>P2에서 TO-프로-3 a 5에 대 한 플롯 하 고 A5중간/높 은 이벤트 모든 세포 파편을 제외를 선택 합니다.</li>
	<li>모든 A5 긍정적인 이벤트를 선택 하 고 a 5에 대 한 FSC 플롯. ApoCells (FSC중간/높은)에서 ApoBDs (FSC낮은)을 구분 합니다. 참고: ApoBDs FACS 기반 접근을 통해 ApoBD 절연을 위한 게이팅, 좋습니다 마지막 단계를 ApoBDs를 선택 하 고 비교를-프로-3 a 5에 모든 이벤트를 선택 하 여. 이렇게 마지막 정렬 게이트 형광 보다는 FSC/SSC 매개 변수를 사용 하면.</li>
	<li>실행 가능한 세포 분석에 대 한 P1을 선택 하 고 두 가지 제어 전략 중 하나를 수행 합니다. 일반 실행 가능한 세포 분석, a 5에 대 한 FSC 플롯 고 따라서 나머지 세포 파편을 제거 하는 모든 FSC중간/높 은 셀을 선택 합니다. 또는, 심층 분석에 대 한 실행 가능한 세포 분리 될 수 있다 a 5에서- 에-프로-3 금감위에 대 한 플롯에 의해 초기 ApoCells. 선택에-프로-3낮은, FSC중간/높은 실행 가능한 세포와 TO-프로-3중간, FSC중간/높 은 A5- 초기 ApoCells.</li>
</ol>

<ol><li>Poon, I. K., Lucas, C. D., Rossi, A. G., Ravichandran, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Apoptotic+cell+clearance%3A+basic+biology+and+therapeutic+potential%5BTitle%5D)+AND+%22Nature+Reviews.+Immunology%22%5BJournal%5D)">Apoptotic cell clearance: basic biology and therapeutic potential.</a> Nature Reviews. Immunology. 14, 166-180 (2014).</li>
<li>Atkin-Smith, G. K., Poon, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disassembly+of+the+Dying%3A+Mechanisms+and+Functions%5BTitle%5D)+AND+%22Trends+in+Cell+Biology%22%5BJournal%5D)">Disassembly of the Dying: Mechanisms and Functions.</a> Trends in Cell Biology. 27, 151-162 (2017).</li>
<li>Tixeira, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defining+the+morphologic+features+and+products+of+cell+disassembly+during+apoptosis%5BTitle%5D)+AND+%22Apoptosis%3A+An+International+Journal+on+Programmed+Cell+Death%22%5BJournal%5D)">Defining the morphologic features and products of cell disassembly during apoptosis.</a> Apoptosis: An International Journal on Programmed Cell Death. 22, 475-477 (2017).</li>
<li>Sebbagh, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Caspase-3-mediated+cleavage+of+ROCK+I+induces+MLC+phosphorylation+and+apoptotic+membrane+blebbing%5BTitle%5D)+AND+%22Nature+Cell+Biology%22%5BJournal%5D)">Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing.</a> Nature Cell Biology. 3, 346-352 (2001).</li>
<li>Coleman, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Membrane+blebbing+during+apoptosis+results+from+caspase-mediated+activation+of+ROCK+I%5BTitle%5D)+AND+%22Nature+Cell+Biology%22%5BJournal%5D)">Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I.</a> Nature Cell Biology. 3, 339-345 (2001).</li>
<li>Atkin-Smith, G. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+mechanism+of+generating+extracellular+vesicles+during+apoptosis+via+a+beads-on-a-string+membrane+structure%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">A novel mechanism of generating extracellular vesicles during apoptosis via a beads-on-a-string membrane structure.</a> Nature Communications. 6, 7439(2015).</li>
<li>Poon, I. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unexpected+link+between+an+antibiotic%2C+pannexin+channels+and+apoptosis%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Unexpected link between an antibiotic, pannexin channels and apoptosis.</a> Nature. 507, 329-334 (2014).</li>
<li>Moss, D. K., Betin, V. M., Malesinski, S. D., Lane, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+role+for+microtubules+in+apoptotic+chromatin+dynamics+and+cellular+fragmentation%5BTitle%5D)+AND+%22Journal+of+Cell+Science%22%5BJournal%5D)">A novel role for microtubules in apoptotic chromatin dynamics and cellular fragmentation.</a> Journal of Cell Science. 119, 2362-2374 (2006).</li>
<li>Kerr, J. F., Wyllie, A. H., Currie, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Apoptosis%3A+a+basic+biological+phenomenon+with+wide-ranging+implications+in+tissue+kinetics%5BTitle%5D)+AND+%22British+journal+of+cancer%22%5BJournal%5D)">Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics.</a> British journal of cancer. 26, 239-257 (1972).</li>
<li>Witasp, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bridge+over+troubled+water%3A+milk+fat+globule+epidermal+growth+factor+8+promotes+human+monocyte-derived+macrophage+clearance+of+non-blebbing+phosphatidylserine-positive+target+cells%5BTitle%5D)+AND+%22Cell+Death+and+Differentiation%22%5BJournal%5D)">Bridge over troubled water: milk fat globule epidermal growth factor 8 promotes human monocyte-derived macrophage clearance of non-blebbing phosphatidylserine-positive target cells.</a> Cell Death and Differentiation. 14, 1063-1065 (2007).</li>
<li>Orlando, K. A., Stone, N. L., Pittman, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rho+kinase+regulates+fragmentation+and+phagocytosis+of+apoptotic+cells%5BTitle%5D)+AND+%22Experimental+Cell+Research%22%5BJournal%5D)">Rho kinase regulates fragmentation and phagocytosis of apoptotic cells.</a> Experimental Cell Research. 312, 5-15 (2006).</li>
<li>Holmgren, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Horizontal+transfer+of+DNA+by+the+uptake+of+apoptotic+bodies%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Horizontal transfer of DNA by the uptake of apoptotic bodies.</a> Blood. 93, 3956-3963 (1999).</li>
<li>Zernecke, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Delivery+of+microRNA-126+by+apoptotic+bodies+induces+CXCL12-dependent+vascular+protection%5BTitle%5D)+AND+%22Science+Signaling%22%5BJournal%5D)">Delivery of microRNA-126 by apoptotic bodies induces CXCL12-dependent vascular protection.</a> Science Signaling. 2, ra81(2009).</li>
<li>Schiller, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autoantigens+are+translocated+into+small+apoptotic+bodies+during+early+stages+of+apoptosis%5BTitle%5D)+AND+%22Cell+Death+and+Differentiation%22%5BJournal%5D)">Autoantigens are translocated into small apoptotic bodies during early stages of apoptosis.</a> Cell Death and Differentiation. 15, 183-191 (2008).</li>
<li>Elamin, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Curcumin+inhibits+the+Sonic+Hedgehog+signaling+pathway+and+triggers+apoptosis+in+medulloblastoma+cells%5BTitle%5D)+AND+%22Molecular+Carcinogenesis%22%5BJournal%5D)">Curcumin inhibits the Sonic Hedgehog signaling pathway and triggers apoptosis in medulloblastoma cells.</a> Molecular Carcinogenesis. 49, 302-314 (2010).</li>
<li>Sagulenko, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AIM2+and+NLRP3+inflammasomes+activate+both+apoptotic+and+pyroptotic+death+pathways+via+ASC%5BTitle%5D)+AND+%22Cell+Death+and+Differentiation%22%5BJournal%5D)">AIM2 and NLRP3 inflammasomes activate both apoptotic and pyroptotic death pathways via ASC.</a> Cell Death and Differentiation. 20, 1149-1160 (2013).</li>
<li>Crescitelli, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+RNA+profiles+in+subpopulations+of+extracellular+vesicles%3A+apoptotic+bodies%2C+microvesicles+and+exosomes%5BTitle%5D)+AND+%22Journal+of+Extracellular+Vesicles%22%5BJournal%5D)">Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes.</a> Journal of Extracellular Vesicles. 2, (2013).</li>
<li>Turiak, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteomic+characterization+of+thymocyte-derived+microvesicles+and+apoptotic+bodies+in+BALB%2Fc+mice%5BTitle%5D)+AND+%22Journal+of+Proteomics%22%5BJournal%5D)">Proteomic characterization of thymocyte-derived microvesicles and apoptotic bodies in BALB/c mice.</a> Journal of Proteomics. 74, 2025-2033 (2011).</li>
<li>Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+assay+for+apoptosis.+Flow+cytometric+detection+of+phosphatidylserine+expression+on+early+apoptotic+cells+using+fluorescein+labelled+Annexin+V%5BTitle%5D)+AND+%22Journal+of+Immunological+Methods%22%5BJournal%5D)">A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V.</a> Journal of Immunological Methods. 184, 39-51 (1995).</li>
<li>Jiang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monitoring+the+progression+of+cell+death+and+the+disassembly+of+dying+cells+by+flow+cytometry%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Monitoring the progression of cell death and the disassembly of dying cells by flow cytometry.</a> Nature Protocols. 11, 655-663 (2016).</li>
<li>Berda-Haddad, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sterile+inflammation+of+endothelial+cell-derived+apoptotic+bodies+is+mediated+by+interleukin-1alpha%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Sterile inflammation of endothelial cell-derived apoptotic bodies is mediated by interleukin-1alpha.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 20684-20689 (2011).</li>
<li>Lleo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shotgun+proteomics%3A+identification+of+unique+protein+profiles+of+apoptotic+bodies+from+biliary+epithelial+cells%5BTitle%5D)+AND+%22Hepatology%22%5BJournal%5D)">Shotgun proteomics: identification of unique protein profiles of apoptotic bodies from biliary epithelial cells.</a> Hepatology. 60, 1314-1323 (2014).</li>
<li>Atkin-Smith, G. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+cell+type-specific+apoptotic+bodies+by+fluorescence-activated+cell+sorting%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Isolation of cell type-specific apoptotic bodies by fluorescence-activated cell sorting.</a> Scientific Reports. 7, 39846(2017).</li>
<li>Jiang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determining+the+contents+and+cell+origins+of+apoptotic+bodies+by+flow+cytometry%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Determining the contents and cell origins of apoptotic bodies by flow cytometry.</a> Scientific Reports. 7, 14444(2017).</li>
</ol>

저자는 공개 없다.

이후 1950 년대에 있는 그것의 초기 설명, apoptosis의 분야 전진 했다 현저 하 게, 저명한 연구 분야를 되 고. 광범위 한 관심과 광범위 한 노력에도 불구 하 고 apoptosis, 특히 ApoBDs의 대형에에서의 하지 되었습니다 잘 공부 적절 한 방법론의 부족. 특히 추적 apoptosis 진행과 동시에 전통적인 cytometry A5/PI 분석 및 ApoBD 격리의 불순물을 사용 하 여 ApoBD 형성에 제한이 포함 됩니다. 최근 접근 방법론 이러한 ?...

Apoptosis, 프로그램 된 세포 죽음의 잘 공부 형태를 생리 적 항상성 유지 하 고 인체1내에서 잠재적으로 유해한 세포를 제거 해야 합니다. Apoptosis 유도 후 apoptotic 세포 (ApoCells) 일련의 형태학 적 변화를 받아야 하 고 ApoBDs 되 나 작은 막 도약 vesicles로 분해 수 있습니다. 전반적으로,이 과정 apoptotic 세포 분해로 알려져 있으며 형태2,3에 따라 3 가지 단계로 나눌 수 있습니다. 단계 1 (플라즈마 멤브레인 blebbing) 풍선 모양의 구조 blebs4,5로 알려진 세포 표면에 형성 특징 이다. 단계 2 (apoptotic 돌출 형성) apoptopodia, 파란색 apoptopodia 및 microtubule 스파이크6,,78등 긴 막 돌출의 형성을 포함합니다. 마지막으로, 3 단계 (ApoBD 형성) apoptotic 돌출 ApoBDs6,9를 생성 하는 ApoCells의 파편을 포함 한다. 이전 연구 결과 apoptotic 세포 클리어런스를 방 조 하 고 세포 통신을 중재에 ApoBDs의 역할을 제안 했다. 예를 들어 ApoBDs에는 ApoCell의 분열 식 세포2,10,11을 둘러싼에 의해 쉽게 제거 될 수 있는 작은 ' 입 크기 ' 조각 생성 수는 제안 합니다. 또한, ApoBDs DNA, RNA 및 단백질, 셀 통신12,,1314를 촉진 하기 위하여 주변 셀에는 인신 매매 수 등 생체의 일련을 항구 수 있습니다. 기능적으로이 프로세스를 조사, apoptosis 유도 및 ApoBD 형성의 유효성 검사 (i), (ii) ApoBDs, 절연 및 (iii) ApoBD 순수성의 확인을 포함 하 여 세 가지 주요 매개 변수를 확인 하는 생명 이다.
이전에, cytometry 등 전자 현미경 검사 법 방법의 수 ApoBDs15,16,,1718apoptosis를 공부 하 고 사용 되었습니다. 그러나, ApoBD 검출 및 정량화는 종종 어려운 또는 간과. 가장 일상적으로 사용 되는 흐름 cytometry 기반 apoptosis 고용 annexin V를 시험 하는 예를 들어, (A5, 단백질은 외부화는 ' 먹고-나 ' 신호 phosphatidylserine (PtdSer))과 핵 산 얼룩 propidium 요오드 화물 (PI)19. 그러나,이 범용 얼룩 조합을 사용 하 여 분석 가정 샘플에서 셀 하위 (가능한 셀, ApoCells 및 회 저 성 세포)의 3 종류가 있습니다. 또한, 비록 apoptosis에 대 한 많은 연구자에 의해 "금 표준"으로 간주, cytometry 분석 실험 및 후속 데이터 분석은 종종 FSC/SSC중간-높 은 이벤트를 선택 하는 초기 제어 단계를 통해 ApoBDs을 제외. 따라서, 우리는 최근에 a 5와 TO-프로-3를 사용 하 여 새로운 흐름 cytometry 분석 결과 개발, caspase 3/7-활성화 pannexin 1 (PANX1)에 의해 선택적으로 촬영 될 수 있는 또 다른 핵 얼룩 채널7,20. Caspase 3 유도 PANX1 활성화 apoptosis의 초기 단계에서 PtdSer 노출 앞로 TO-프로-3 차동 apoptotic 및 회 저 성 세포를 얼룩. 또한, 전략 게이팅 우리의 소설과 결합 된이 접근 데이터 분석 중 모든 획득된 이벤트를 포함 하 고 그 결과, 6 셀/입자 하위 집합 식별 됩니다, 포함 하 여: (i) 가능한 셀 (FSC/SSC중간/높은, A5낮은 , TO-프로-3낮은), (ii) A5- 초기 ApoCells (FSC/SSC중간/높은, A5낮은,중간에-프로-3), (iii) A5+ ApoCells (FSC/SSC중간/높은, A5높은 ,중간에-프로-3), (iv) 회 저 성 세포 또는 늦은 ApoCells (FSC/SSC중간/높은 A5높은,높은TO-프로-3), (v) ApoBDs (FSC/SSC낮은, A5중간, TO-프로-3낮은 중간 /), 그리고 (vi) 파편 (FSC/SSC낮은,낮은A5, TO-프로-3낮은)20. 우리의 접근 방식은 모든 셀/입자 하위 집합 및, 더 중요 한 것은, 세포와 파편20에서 ApoBDs의 분리 분석의 중요성을 강조 한다. 따라서,이 방법은 apoptosis와 ApoBD 형성의 유도 동시에 확인 하는 효율적인 기법을 보여 줍니다.
전통적으로, ApoBDs는 ApoBDs 셀 또는 밀도에 따라 다른 세포 외 소포에서 분리 될 수 있다 그것에 의하여 차별 원심 분리 방식의 다양 한 격리 되었습니다. 그러나, 이러한 원심 분리 방법은 종종 낮은 ApoBD 순수성, 샘플 순도 및 셀 형식 관련 ApoBDs17,,2122분리를 확인 정량화 단계의 부족에 의해 제한 됩니다. 따라서, 우리 최근 두 가지 방법, FACS 기반 및 새로운 차등 원심 분리-기반 접근 apoptosis와 샘플 순도23의 유도 확인 하기 위해 우리의 이전 설립된 흐름 cytometry 방법으로 결합 될 수 있는 개발. FACS 기반 접근을 통해 ApoBD 절연 99% 순도, 최대 ApoBDs을 풍요롭게 수 있습니다 하 고 혼합된 셀 인구, 조직 샘플 및 체액23에서 ApoBDs을 셀 유형 특정 항 체의 다양 한 결합 될 수 있다. 또한, 우리의 개정된 차등 원심 분리 방식을 보여줍니다 ApoBDs를 분리 하는 효율적인 방법 > 90% 순도23.
이 종이, 우리가 자세히 설명 apoptosis 유도, 유효성을 검사 하 고 검색 하 고 ApoBD 형성 계량 우리의 실험 절차. FACS 기반 및 차등 원심 기반 방법을 사용 하 여 ApoBD 절연 워크플로 또한 정교 하 고 비교. 대표적인 데이터 설명된 방법론 미래 ApoBD 연구에 대 한 효과적인 첨단 도구를 제공 합니다 보여줍니다.

<table><tbody><tr><td>Cells e.g. cultured human THP-1 monocytes (clone number: TIB-202)</td><td>ATCC</td><td>-</td><td></td></tr><tr><td>RPMI 1640 medium</td><td>Life Technologies</td><td>22400-089</td><td></td></tr><tr><td>Penicillin-streptomycin mixture</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>FSC</td><td>Gibco</td><td>10099-141</td><td></td></tr><tr><td>1x PBS</td><td>-</td><td>-</td><td></td></tr><tr><td>Annexin V FITC</td><td>BD Bioscience</td><td>-</td><td></td></tr><tr><td>TO-PRO-3 iodide</td><td>Life Technologies</td><td>T3605</td><td>TO-PRO-3 may cause skin, eye and respiratory irration. Avoid direct contact.</td></tr><tr><td>10x Annexin V binding buffer</td><td>BD Bioscience</td><td>556454</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>1001710526</td><td></td></tr><tr><td>Centrifuge tube (15 mL)</td><td>Cellstar</td><td>188271</td><td></td></tr><tr><td>Microcentrifuge tube (1.5 mL)</td><td>Sarstedt</td><td>72.690.001</td><td></td></tr><tr><td>Tissue culture incubator (37 &amp;deg;C, 5% CO2)</td><td>-</td><td>-</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td>392932</td><td></td></tr><tr><td>FACS ARIA III Flow cytometer, configured with two lasers for FITC and APC detection</td><td>BD Bioscience</td><td>-</td><td></td></tr><tr><td>FACS Canto II Flow cytometer, configured with two lasers for FITC and APC detection</td><td>BD Bioscience</td><td>-</td><td></td></tr><tr><td>FACS Diva 6.1.1 software</td><td>BD Bioscience</td><td>-</td><td></td></tr><tr><td>FlowJo 8.8.6 software</td><td>-</td><td>-</td><td></td></tr><tr><td>&amp;nbsp;UV Stratalinker 1800</td><td>Stratagene</td><td>-</td><td></td></tr></tbody></table>

detection and isolation of apoptotic bodies to high purity

Apoptotic 시체 (ApoBDs), microvesicles 및 exosomes ApoBDs 가장 큰 종류 중 하나 되 고 함께 세포 외 기 가족의 주요 회원입니다. 그것은 ApoBDs 밀매 생체 세포 통신으로 셀 정리 도움이 될 수 있습니다 제안 되었습니다. 식별 및 ApoBDs의 격리에 사용 하는 기존의 접근은 종종 정확한 정량화 및 낮은 샘플 순도의 부족에 의해 제한 됩니다. 여기, 우리는 apoptosis 유도 확인 하 고, ApoBD 형성, 검증 순도를 ApoBDs를 분리 하는 워크플로 설명 합니다. 우리 또한 약술 하 고 형광 활성화 된 세포 (FACS) 정렬 및 ApoBDs을 기반으로 하는 차등 원심 분리 접근 비교 것입니다. 또한, 고립 된 ApoBDs의 순도 확인 될 것 이다 사용 하는 이전 교류 cytometry 기반 얼룩이 지 고 분석 방법 확립. 기술된 접근을 사용 하 여 함께 찍은 THP 1 monocyte apoptosis와 apoptotic 세포 분해 했다 유도 검증, 그리고 THP 1 monocytes에서 생성 된 ApoBD 97-99%의 순수성에 고립 되었다.

여기에 설명 된 절차를 사용 하 여, THP 1 monocyte apoptosis 유도 되었다 그리고 ApoBDs 탐지 되 고 고립 FACS 기반 또는 차등 원심 분리 방식 (그림 1)를 통해. 첫째, apoptosis UV 방사선에 의해 유도 되었다 및 샘플 셀 apoptotic 형태학, blebbing, apoptotic 막 돌출 형성 및 ApoBDs6의 세대를 포함 하 여 전시 하는 때 외피의 2-3 h 후 수집 된. TO-프로-3와 A5 ?...

Watch this Scientific Journal Video about Detection and Isolation of Apoptotic Bodies to High Purity at JoVE.com

Detection and Isolation of Apoptotic Bodies to High Purity

교류 cytometry 또는 차등 원심 분리를 사용 하 여 워크플로 검색, 계량 및 apoptotic 시체는 apoptotic 샘플 고 순도 분리 개발 된다.

검색 및 Apoptotic 몸 높은 순도 절연

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. ...

detection-and-isolation-of-apoptotic-bodies-to-high-purity

Research

JoVE Journal

Biochemistry

10.6K 조회수.  La Trobe University. 교류 cytometry 또는 차등 원심 분리를 사용 하 여 워크플로 검색, 계량 및 apoptotic 시체는 apoptotic 샘플 고 순도 분리 개발 된다.

비디오: Detection and Isolation of Apoptotic Bodies to High Purity

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

새로 형성 된 유방암 줄기 세포와 같은 세포 Apoptosis 반전 후 흐름 Cytometric 감지

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

연구

암 연구학

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes 8, 16. If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades 14. Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer's, Huntington's, and Multiple Sclerosis 4, 14, 3, 11, 7.
In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures 15, 5, following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience) 15, 5, and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation (e.g.6, 10). We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein) 13, 12. Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis 9. We used a bi-functional caspase-3 substrate containing a DEVD (Asp-Glu-Val-Asp) caspase-3 recognition subunit and a DNA-binding dye 2. The substrate quickly enters the cytoplasm where it is cleaved by intracellular caspase-3. The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time 1, 10. In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques.

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depola

Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.

모니터링 죽습 Caspase - 3 활동 및 칼륨 - 유도 막 탈분극 다음 라이브 세포 이미징 및 Cleaveable 형광 염색 기판을 사용하여 불후의 Oligodendroglial 세포의 Apoptosis

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a ...

신경과학

Flow Cytometric Analysis of Apoptotic Biomarkers in Actinomycin D-Treated SiHa Cervical Cancer Cells

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V and mitochondrial membrane potential) and late biomarkers (caspases 3 and 7, and DNA damage) of apoptosis were measured in experimental and control cultures. Cultures were incubated for 24 hours in a humidified incubator at 37 &#176;C with 5% CO2. The cells were then detached using trypsin and enumerated using a flow cytometric cell count assay. Cells were further analyzed for apoptosis using an Annexin V assay, a mitochondrial electrochemical transmembrane potential assay, a caspase 3/7 assay, and a DNA damage assay. This article provides an overview of apoptosis and traditional flow cytometry, and elaborates flow cytometric protocols for processing and analyzing SiHa cells. The results describe positive, negative, and sub-optimal experimental data. Also discussed are interpretation and caveats in performing flow cytometric analysis of apoptosis using this analytical platform. Flow cytometric analysis provides an accurate measurement of early and late biomarkers for apoptosis.

Apoptosis can be characterized by flow cytometric analysis of early and late apoptotic biomarkers. The cervical cancer cell line, SiHa, was analyzed for apoptosis biomarkers after treatment with Actinomycin D using a benchtop flow cytometer.

Actinomycin D-Treated SiHa 자궁경부암 세포에서 세포사멸 바이오마커의 유세포 분석

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V ...

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

시상 하부 세포 모델에서 세포 사멸을 결정하는 카스파 다중 분석의 사용

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators and executioners of apoptosis, the most studied form of programmed cell death. Apoptosis occurs during developmental processes and is a necessary event in tissue homeostasis. Pyroptosis is another form of cell death that utilizes caspases and is a critical process in activating the immune system through the activation of the inflammasome, which results in the release of members of the interleukin-1 (IL-1) family. To assess caspase activity, target substrates can be assessed. However, sensitivity can be an issue when examining single cells or low-level activity. We demonstrate how a fluorogenic substrate can be used with a population-based assay or single-cell assay by flow cytometry. With proper controls, different amino acid sequences can be used to identify which caspases are active. Using these assays, the simultaneous loss of the inhibitors of apoptosis proteins upon tumor necrosis factor (TNF) stimulation has been identified, which primarily induces apoptosis in macrophages rather than other forms of cell death.

The present protocol describes two methods to measure caspase activity through a fluorogenic substrate using flow cytometry or a spectrofluorometer.

형광 분석 또는 유세포분석을 사용한 카스파제 활성 측정

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators ...

생화학

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability1,2. The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells3. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence 1,2,9. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment10. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios &lt;0.5) showing the highest occurrence10. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (&lt; 5% events with cytoplasmic PI staining).

An accurate method for the assessment of cell death is described. The protocol improves upon conventional Annexin V/ propidium iodide (PI) protocols, which display up to 40% false- positive events in cell lines and primary cells from a broad range of animal models.

세포 죽음의 정확한 평가에 대한 수정 Annexin V / Propidium 이우의 Apoptosis 분석

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely ...

생물학

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

단일 세포 형광 현미경 검사 법에 의해 Efferocytosis의 정량화

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

면역학 및 감염병학

Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution of inflammation. Accumulation of apoptotic debris in the body may trigger chronic inflammatory responses that lead to systemic autoimmune diseases over time. Impaired apoptotic cell clearance has been implicated in a variety of autoimmune diseases. Apoptotic clearance is a complex process rarely detected under physiological conditions. It involves abundant surface receptors and signaling molecules. Studying the process of apoptotic cell clearance provides insightful molecular mechanisms and subsequent biological responses, which may lead to the development of new therapeutics. Here, we describe protocols for the induction of apoptotic thymocytes, the preparation of peritoneal macrophages, and the analysis of apoptotic cell clearance by flow cytometry and microscopy. All cells will undergo apoptosis at a certain stage, and many residential and circulating cells can uptake apoptotic debris. Therefore, the protocol described here can be used in many applications to characterize apoptotic cell binding and ingestion by many other cell types.

Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

대 식 세포에의 한 사 멸 티 모 세포 침 몰의 실험 분석

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution ...

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

추적 아나스타시스, 세포 사멸을 취소 세포 생존 현상에 대한 전략

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

세포 내 이웃 셀을받은 사멸 세포 죽음과의 상호 작용에 의해 실행 가능한 세포에서 유도 된 이벤트를 시그널링의 식별

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...