이 연구는 자연 과학 및 캐나다 (NSERC) 연구 부여 공학 의회와 DRB하는 앨버타 농업 기금 컨소시엄 부여에 의해 지원되었다. AMR은 NSERC 베니 캐나다 대학원 장학금, 앨버타 교육 assistantship와 퀸 엘리자베스 II 대학원 장학금의 대학을 통해 지원됩니다.

이 절차는 500 μL siliconized 튜브 또는 6 ML의 폴리스티렌 왕복 하단 외과 튜브에서 수행할 수 있습니다. 다른 절차와 함께 생존 분석을 수행하면 후자가 일반적으로 사용됩니다. 주어진 모든 볼륨 6 ML의 폴리스티렌 왕복 하단 외과 튜브위한 것입니다. 500 μL siliconized 튜브의 경우, 1 / 5 모든 볼륨을 줄일 수 있습니다. 유동세포계측법 분석을위한 최적의 농도는 200 μL 볼륨 당 2-4 X 10 6 세포이다. 셀 손실이 절차에서 발생할 수 있습니다 때문에 우리는 각 예제에서는 프로 시저의 시작 부분에 4 X 10 6 세포로 구성하는 것이 좋습니다. 1. 셀 준비 <ol><li> 하베스트 세포는 - 해당 셀 라인 기본 셀 isolations에 대한 구체적인 절차를 참조하십시오. </li><li> 10 분 335 X g에서 샘플을 원심 분리기 및 뜨는을 가만히 따르다. </li><li> 2 ML 1 X 인산의 Resuspend 전지 (PBS) 호수 버퍼 - / - (없음 칼슘, 마그네슘 없음). </li><li> 10 분 335 X g에서 샘플을 원심 분리기 및 뜨는을 가만히 따르다. </li><li> 1 ML 일 X Annexin V 바인딩을 버퍼에 셀을 Resuspend. </li><li> 10 분 335 X g에서 샘플을 원심 분리기 및 뜨는을 가만히 따르다. </li><li> 100 μL 1 X Annexin V 바인딩을 버퍼에 Resuspend 세포. </li></ol> 2. Annexin V / PI의 응용 프로그램 스테인 <ol><li> 제조 업체의 권고에 따라 Annexin V를 추가합니다 [예 : 5 μL Annexin V 알렉사 플루어 488 (분자 프로브, A13201)]. </li><li> 실온에서 15 분 어둠 속에 튜브를 품어. </li><li> 각 반응 관 1 X Annexin V 바인딩 버퍼 100 μL를 추가합니다. 각각의 튜브에 약 200 μL이 있어야합니다. </li><li> 1 X Annexin V 구속력 버퍼 (X Annexin V 구속력 버퍼 9 μL 1 즉, 1 μL PI)에 1시 10분 희석되었습니다 PI 4 μL (시그마, 고양이 # P - 4864 - 10ML)를 추가합니다. 이것은 각각의 샘플 2 μg / ML의 최종 PI 농도를 얻을 수 있습니다. </li><li> 실온에서 15 분 어둠 속에 튜브를 품어. </li><li> 세포를 씻어 500 μL 1 X Annexin V 바인딩 버퍼를 추가합니다. </li><li> 10 분 335 X g에서 샘플을 원심 분리기 및 뜨는을 가만히 따르다. </li><li> 500 μL 1 X Annexin V 바인딩 버퍼와 1 %의 포름 알데히드 (정착액) 솔루션을 만들려면 500 μL 2 % 포름 알데히드에 Resuspend 세포. 부드러운 flicking으로 튜브를 섞는다. </li><li> 10 분 얼음에 샘플을 수정. 또한, 샘플은 4 박 저장할 수 있습니다 ° C를 어둠에 약해. 후자의 방법이 선택되면, Annexin V / PI (보상 제어 포함)으로 표시 모든 튜브에 걸쳐 일관되게해야합니다. </li><li> 각 샘플과 flicking에 의해 부드럽게 믹스 - / - 1 ML 1 X PBS를 추가합니다. </li><li> 8 분에 425 X g에서 튜브를 원심 분리기 및 뜨는을 가만히 따르다. </li><li> 단계 2.10와 2.11를 반복합니다. </li><li> 튜브를 flicking하여 Resuspend 펠릿. </li><li> 50 μg / ML의 최종 농도를 제공 1:100 희석 RNase A (시그마, R4642) 16 μL를 추가합니다. 37 15 분 품어 ° C. </li><li> / - - 1 ML 1 X PBS를 추가하고 flicking으로 가볍게 섞는다. </li><li> 8 분에 425 X g에서 튜브를 원심 분리기. </li><li> 샘플 지금 분석 준비가 된 것입니다. Annexin / PI 염색법 다른 절차를 병렬로 진행되는 경우 또는 샘플이 이후 얼룩 단계에 사용할 수 있습니다. </li></ol> 3. 대표 결과 : 거짓 - 긍정적인 PI의 얼룩의 학위를 가지고 다양한 세포 종류와 세포 라인의 예는 그림 1에 표시됩니다. (그림 2B) RNase의 치료 안 세포에 비해, 거짓 - 긍정적인 이벤트의 수가 크게 감소 거짓 - 긍정적인 이벤트에 대한 계정으로, 세포가 수정되었습니다 다음이 결과를 RNase A. 취급. 그림 2C는 RNase 처리 없었다 세포에 비해 RNase 치료를 받았습니다 세포의 대표 이미지를 보여줍니다. 그림 3은 고정 및 RNase 처리 단계를 통해 수정된 Annexin V / PI 프로토콜, 허위 - 긍정적인 얼룩 이벤트의 수를 제한하여 기존 프로토콜에 따라 개선 방법을 보여줍니다. <img src="/files/ftp_upload/2597/2597fig1.jpg" alt="그림 1" /> 그림 1. 기존 propidium의 요오드화물 얼룩 프로토콜은 거짓 긍정적인 사건의 상당수를 얻으실 수 있습니다. 우리는 이전에 동물 모델의 광범위한 걸쳐뿐만 아니라 세포 라인 10의 다양한 기본 세포를 통해 잘못된 긍정적인 얼룩의 정도를 평가합니다. 대표 이미지는 세 독특한 세포 집단 (- RAW macrophages과 두 가지 기본 세포 - murine 골수 macrophages (BMM)와 금붕어 기본 신장 macrophages (PKM) 1 일반적으로 사용되는 세포주)에서 잘못된 긍정적인 얼룩의 범위를 표시합니다. 진정한 긍정적인 이벤트가 핵 구획 내에서 PI와 DRAQ5 사이의 예상 공동 현지화 (노란색 영역 PI와 DRAQ5 사이 colocalization를 묘사)를 표시합니다. DRAQ5 매우 membra입니다NE 투과 염색되는 살고 죽은 세포 10,11,12 모두 정확하게 얼룩 핵 구획. colocalization DRAQ5보다 쉽게​​ 시각 결정을위한 얼룩은 녹색 pseudocolour을 받았다. <img src="/files/ftp_upload/2597/2597fig2.jpg" alt="그림 2" /> 그림 2. PI 핵 얼룩의 정확성을 향상. (A) 우리는 DRAQ5을 잘 특징 핵 얼룩의 숫자 (BrdU, 7 - AAD 및 DAPI)의 유사성의 정도에 따라 PI 핵 얼룩의 정확성을 평가. BrdU는 proliferating 세포의 DNA에 통합하고, 같은 오직 핵 구획 내에 착색하는 합성 뉴클레오입니다. 7 - AAD는 손상 플라즈마 막을 통과하지 못하면, PI와 비슷한 높은 DNA에 대한 친화력과을 가지고 있습니다. DAPI는 DNA에 대한 강한 친화력을 가지고 있으며, 가장 일반적으로 사용되는 핵은 형광 현미경에 얼룩 있습니다. 유사성의 정도가 정확하게 RAW 264.7 macrophages의 세포 핵을 얼룩이 자신의 능력을 강조, BrdU, 7 - AAD, DAPI와 DRAQ5 사이&gt; 99%되었다. 대조적으로, 기존의 프로토콜을 기반으로 세포질 PI의 얼룩은 DRAQ5에 상대적으로 얼룩의 %의 유사성에 표시된 감소로 연결됩니다. murine 골수 macrophages (BMM)와 금붕어 기본 신장 macrophages (PKM) (B)와 마찬가지로 결과는 테스트를 두 가지 기본 세포에서 관찰됩니다. 50 μg / ML RNase 얼룩 절차 내의 특정 단계에서의 결합이 아닌 핵 PI의 얼룩을 제거하고 크게 DRAQ5 얼룩에 상대적 유사성의 정도를 증가시킵니다. (C) 기본 신장 macrophages의 대표 이미지와 RNase 처리없이 세포에 대한 유사성의 정도를 보여줍니다. 트리 트먼트 차이보다 쉽게​​ 시각 결정을위한, DRAQ5는 녹색을 받았다. 노란색 영역은 BrdU와 DRAQ5, 그리고 PI와 DRAQ5 사이 colocalization을 묘사. <img src="/files/ftp_upload/2597/2597fig3.jpg" alt="그림 3" /> 그림 3. 수정 Annexin V / PI 크게 잘못된 apoptosis / 괴사성 이벤트를 줄여줍니다. 기본 금붕어 신장 macrophages (PKM)는 종래와 수정 Annexin V / PI 프로토콜 물들일되었습니다. Scatterplots는 Annexin V / PI는 금붕어 PKM에 얼룩 묘사. 왼쪽 scatterplot는 PKM 세포의 일반적인 Annexin V / PI 염색법을 (NO RNase)를 보여줍니다. 오른쪽 scatterplot은 수정 Annexin V / PI 프로토콜 (RNase)를 물들일 PKM 세포를 보여줍니다. 거짓 긍정적인 얼룩의 제거에 의한 PI의 얼룩이 표시된 감소 RNase 결과뿐만 아니라. PKM 세포 그런 다음 서로 다른 문화 - 초기 progenitors 이내에 인구 (EP), monocytes (모노)와 성숙 macrophages (매트 MAC)를 기반으로 분석했다. 거짓 긍정적인 사건의 제거로 인해 긍정적인 PI 이벤트의 수가 감소가 작은 초기 전구 세포보다 큰 성숙 세포 대식 세포의 세포에 더 많은 발음입니다. 기존의 프로토콜에 따라 결론이 잘못보다 성숙한 대식 세포의 하위 집합에 대한 세포의 죽음에 긍정적인 상관 관계를 제안 것입니다.

<ol>
	<li>Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+assay+for+apoptosis.+Flow+cytometric+detection+of+phosphatidylserine+expression+on+early+apoptotic+cells+using+fluorescein+labeled+Annexin+V.">A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V.</a> J Immunol Methods. 184, 39-51 (1995).</li><li>Vermes, I., Haanen, C., Reutelingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+of+apoptotic+cell+death.">Flow cytometry of apoptotic cell death.</a> J Immunol Methods. 243, 167-190 (2000).</li><li>Cornelissen, M., Philippe, J., De Sitter, S., De Ridder, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annexin+V+expression+in+apoptotic+peripheral+blood+lymphocytes:+An+electron+microscopic+evaluation.">Annexin V expression in apoptotic peripheral blood lymphocytes: An electron microscopic evaluation.</a> Apoptosis. 7, 41-47 (2002).</li><li>Fried, J., Perez, A. G., Clarkson, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytofluorometric+analysis+of+cell+cycle+distributions+using+propidium+iodide.+Properties+of+the+method+and+mathematical+analysis+of+the+data.">Flow cytofluorometric analysis of cell cycle distributions using propidium iodide. Properties of the method and mathematical analysis of the data.</a> J Cell Biol. 71, 172-181 (1976).</li><li>Bacso, Z., Everson, R. B., Eliason, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA+of+Annexin+V-binding+apoptotic+cells+is+highly+fragmented.">The DNA of Annexin V-binding apoptotic cells is highly fragmented.</a> Cancer Res. 60, 4623-4628 (2000).</li><li>Darzynkiewicz, Z., Bruno, S., Del Bino, G., Gorczyca, W., Hotz, M. A., Lassota, P., Traganos, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+of+apoptotic+cells+measured+by+flow+cytometry.">Features of apoptotic cells measured by flow cytometry.</a> Cytometry. 13, 795-808 (1992).</li><li>Kroemer, G., Dallaporta, B., Resche-Rigon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+death/life+regulator+in+apoptosis+and+necrosis.">The mitochondrial death/life regulator in apoptosis and necrosis.</a> Annu. Rev. Physiol. 60, 619-642 (1998).</li><li>Denecker, G., Vercammen, D., Declercq, W., Vandenabeele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+and+necrotic+cell+death+induced+by+death+domain+receptors.">Apoptotic and necrotic cell death induced by death domain receptors.</a> Cell Mol. Life Sci. 58, 356-370 (2001).</li><li>Faleiro, L., Lazebnik, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspases+disrupt+the+nuclear-cytoplasmic+barrier.">Caspases disrupt the nuclear-cytoplasmic barrier.</a> J Cell Biol. 151, 951-959 (2000).</li><li>Rieger, A. M., Hall, B. E., Luong, L. T., Schang, L. M., Barreda, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conventional+apoptosis+assays+using+propidium+iodide+generate+a+significant+number+of+false+positives+that+prevent+accurate+assessment+of+cell+death.">Conventional apoptosis assays using propidium iodide generate a significant number of false positives that prevent accurate assessment of cell death.</a> J Immunol Methods. 358, 81-92 (2010).</li><li>Edward, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+Use+of+DNA-specific+anthraquinone+dyes+to+directly+reveal+cytoplasmic+and+nuclear+boundaries+in+live+and+fixed+cells.">Minireview: Use of DNA-specific anthraquinone dyes to directly reveal cytoplasmic and nuclear boundaries in live and fixed cells.</a> Mol. Cells. 27, 391-396 (2009).</li><li>Njoh, K. L., Patterson, L. H., Zloh, M., Wiltshire, M., Fisher, J., Chappell, S., Ameer-Beg, S., Bai, Y., Matthews, D., Errington, R. J., Smith, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+analysis+of+the+DNA+targeting+bisalkylaminoanthraquinone+DRAQ5+in+intact+living+cells.">Spectral analysis of the DNA targeting bisalkylaminoanthraquinone DRAQ5 in intact living cells.</a> Cytometry Part A. 69, 805-814 (2006).</li></ol>

관심 없음 충돌 선언하지 않습니다.

Annexin V / PI 프로토콜 여기서 제시하는 것은 기존 프로토콜의 수정된 버전이며 계정으로 또한 PI에 대한 높은 친화력을 가지고있는 세포질에서 RNA의 존재를 걸립니다. 얼룩 절차 후반 1 %의 포름 알데히드 고정 단계 다음 RNase (50 μg / ML)의 소개 원자력 PI의 얼룩의 정확도를 크게 향상시킵니다. 대한 부정적인 영향은 핵 PI의 얼룩이나 플라즈마 막 Annexin V의 얼룩에서 관찰되지 않습니다. RNase 치료없이 얼룩 PI 결과의 최대 40 %가 하류 결론 10 잠재적으로 중요하고 부정적인 영향에 이르게 거짓 긍정적인 행사로 이어질 수 있습니다. 우리 수정 Annexin V / PI 염색법 프로토콜은 간단하고 세포 유형 (기본 세포의 광범위한 범위에서 효과적으로 사용되었습니다 murine 골수 macrophages 및 splenocytes, 돼지 폐 재류 세포, 폐 폐포 macrophages, mesenteric 림프 노드 격리, 말초 혈액 leukocytes , splenocytes, 닭고기 배반엽 세포, 금붕어 기본 신장 macrophages, 잉어 말초 혈액 leukocytes, 셀 라인 : RAW 264.7 macrophages, Jurkat T 세포, 돼지 3D4/31 macrophages, CCL - 71 금붕어 지느러미 섬유아 세포, 3B11 메기의 B 세포 10). 세포가 differentially 차 전지는 일반적으로 잘못된 긍정적인 이벤트 10 큰 숫자를 가지고와 잘못된 긍정적인 PI의 얼룩에 의해 영향됩니다하는 것이 중요합니다. 이 세포 집단 간의 차이를 거짓 긍정적인 PI의 얼룩은 부분적으로 셀 크기의 차이로 표시될 수 있습니다뿐만 아니라 RNA 내용의 차이에서 발생할 수 있습니다. 이 절차의 예, 결합은 다른 사람 사이에 활용할 실험 시스템, genotoxic 스트레스를 겪고 세포, 세포주기 체포 등 thymidine 또는 hydroxyurea와 같은 약물, virally 감염된 세포 및 발달 진행 배아 세포 연구로 치료 세포에 특히 관련으로 세포 RN​​A 합성의 이산 변화이 특징입니다.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>1 x PBS-/-</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1 x Annexin V Binding Buffer</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>556454</td>
<td>&nbsp;</td>
<tr>
<td>Annexin V-Alexa Fluor 488</td>
<td>&nbsp;</td>
<td>Molecular Probes (Invitrogen)</td>
<td>A13201</td>
<td>&nbsp;</td>
<tr>
<td>Propidium iodide (PI)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P4864</td>
<td>1 mg/mL in H2O</td>
</tr>
<tr>
<td>2% Formaldehyde</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>F1268</td>
<td>&nbsp;</td>
<tr>
<td>RNase A from bovine pancreas</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>R4642</td>
<td>&nbsp;</td>
<tr>
<td>DRAQ5</td>
<td>&nbsp;</td>
<td>Biostatus</td>
<td>DR50050</td>
<td>Dilute 1:60 in 1 x PBS-/-</td>
</tr>		</tbody>
		</table>
		</div>

modified annexin v/propidium iodide apoptosis assay for accurate assessment of cell death

세포 apoptosis의 연구는 크게 흐름 cytometry 기반 방법의 도입 이후 영향되었습니다. Propidium 요오드화물의 (PI)가 널리 세포가 플라즈마 막 무결성 및 투자율의 1,2의 차이를 통해 가능한, apoptotic 또는 괴사성 있는지 확인하기 위해 Annexin V와 함께 사용됩니다. Annexin V / PI 프로토콜은 apoptotic 세포에게 3 공부를 위해 일반적으로 사용되는 방법입니다. 그것이 세포가 4,5 생활 염료를 제외하기 위해 능력을 바탕으로, 경제적 안정과 세포 생존의 좋은 지표이기 때문에 PI는 더 자주 다른 핵 얼룩보다 사용됩니다. 셀 입력 PI의 능력은 막의 투자율에 의존적일 수, PI는 손상 플라즈마 막 1,2,6의 존재로 인해 거주하거나 초기 apoptotic 세포를 얼룩이되지 않습니다. 늦은 apoptotic 및 괴사성 세포에서 플라즈마와 핵 세포막의 무결성은 PI가 세포막을 통과 핵산에 사이에 끼어 넣다, 붉은 형광 1,2,9을 표시할 수 있도록, 7,8을 감소시킵니다. 불행히도, 우리는 그런 기존 Annexin V / PI 프로토콜은 세포질 구획 10 시간 RNA의 PI의 얼룩과 관련된 잘못된 긍정적인 사건의 상당수 (최대 40 %)에 리드 찾습니다. 가장 높은 발생 10보기 : (세포질 비율 &lt;0.5 핵) 기본 세포와 동물 모델의 광범위한 셀 라인은 큰 세포와 영향을받습니다. 여기에, 우리는 기존의 방법에 비해 세포 죽음의 평가에 대한 상당한 개선을 제공하는 수정 Annexin V / PI 방법을 보여줍니다. 이 프로토콜은 다음과 같은 얼룩 세포에 RNase의 진입을 촉진하기 위해 고정 셀 동안 세포 투과성의 변화에​​ 활용합니다. 타이밍 및 RNase의 농도 모두 세포질 RNA의 제거를 위해 최적화되었습니다. 그 결과 종래의 Annexin V / PI 프로토콜 (세포질 PI의 얼룩과 &lt;5 % 이벤트)을 통해 상당한 개선이다.

Watch this Scientific Journal Video about Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death at JoVE.com

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

세포 죽음의 평가에 대한 정확한 방법을 설명합니다. 프로토콜은 세포 라인과 동물 모델의 광범위한 범위에서 기본 세포 40 % 허위 - 긍정적인 이벤트까지 표시 기존 Annexin V / propidium 요오드화물 (PI) 프로토콜에 따라이 향상됩니다.

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 ...

modified-annexin-vpropidium-iodide-apoptosis-assay-for-accurate

Research

JoVE Journal

Biology

102.4K 조회수.  University of Alberta. 세포 죽음의 평가에 대한 정확한 방법을 설명합니다. 프로토콜은 세포 라인과 동물 모델의 광범위한 범위에서 기본 세포 40 % 허위 - 긍정적인 이벤트까지 표시 기존 Annexin V / propidium 요오드화물 (PI) 프로토콜에 따라이 향상됩니다.

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

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

Cardiogenic 유도 효율을 Quantifying에 대한 수정 마우스 배아의 줄기 세포 기반 어세이

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

연구

생물학

A &#946;-glucuronidase (GUS) Based Cell Death Assay

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative positive or negative regulators of cell death. In this system, N. benthamiana leaves are co-infiltrated with a 35S driven expression cassette containing the gene to be analyzed, and the GUS vector pCAMBIA 2301 using Agrobacterium strain LBA4404 as a vehicle. Because live cells are required for GUS expression to occur, loss of GUS activity is expected when this marker gene is co-expressed with positive regulators of cell death. Equally, increased GUS activity is observed when anti-apoptotic genes are used compared to the vector control. As shown below, we have successfully used this system in our lab to analyze both pro- and anti-death players. These include the plant anti-apoptotic Bcl-2 Associated athanoGene (BAG) family, as well as, known mammalian inducers of cell death, such as BAX. Additionally, we have used this system to analyze the death function of specific truncations within proteins, which could provide clues on the possible post-translational modification/activation of these proteins. Here, we present a rapid and sensitive plant based method, as an initial step in investigating the death function of specific genes.

A &amp;#946;-glucuronidase GUS Based Cell Death Assay

Programmed cell death assays commonly used in mammalian systems such as DNA laddering or TUNEL assays, are often difficult to reproduce in plants. In combination with a GUS reporter system, we propose a rapid, plant based transient assay to analyze the potential death properties of specific genes.

β - glucuronidase (거스)을 기반으로 세포를 파괴 분석

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

시토크롬의 세포질 Microinjection 의한 Apoptosis의 활성화 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

태아의 프로그래밍 셀 죽음을 감지 할 LysoTracker의 이용 및 배아 줄기 세포를 차별화

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

모자이크 성인의 라이브 이미징 토마토 / GFP-FLP / FRT 방법<em&gt; 초파리</em&gt; 광 수용체 (photoreceptor)

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

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 ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Polysom​​e 프로파일 링 포유 동물 세포에 선택적 mRNA의 번역 평가

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and pathological roles, there is need of high-throughput screening (HTS) assays to identify GJ modulators in drug discovery and toxicology assays. A novel iodide-yellow fluorescent protein-gap junction-intercellular communication (I-YFP-GJIC) assay fulfills this need. It is a cell-based assay including acceptor and donor cells that are engineered to stably express a yellow fluorescent protein (YFP) variant, whose fluorescence is sensitively quenched by iodide, or SLC26A4, an iodide transporter, respectively. When iodide is added to a mixed culture of the two cell types, they enter the donor cells via the SLC26A4 transporter and diffuse to the adjacent acceptor cells via GJs where they quench the YFP fluorescence. YFP fluorescence is measured well by well in a kinetic mode. The YFP quenching rate reflects GJ activity. The assay is reliable and rapid enough to be used for HTS. The protocol for the I-YFP-GJIC assay using the LN215 cells, human glioma cells, is described.

Here, we present a protocol for a novel gap junction intercellular communication assay designed for the high-throughput screening of gap junction-modulating chemicals for drug discovery and toxicological assessment.

요오드 화물-노랑 형광 단백질-간격 접속점 세포 통신 분석 결과

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

성인 근육 줄기 세포 기능 전 비보의 평가를위한 단일 근섬유 배양 분석

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous research indicated that multiple pathways could mediate the spontaneous death of neutrophils. A cocktail was developed by simultaneously targeting these pathways, caspases-lysosomal membrane permeabilization-oxidant-necroptosis inhibition plus granulocyte colony-stimulating factor (CLON-G), which prolonged the neutrophil lifespan to greater than 5 days without significantly compromising the neutrophil function. Concurrently, a reliable and stable protocol for assessing and evaluating neutrophil death was also developed. In this work, we show that CLON-G can prolong the neutrophil lifespan in vitro to more than 5 days, and we exhibit the lengthening of the neutrophil lifespan with FACS and confocal fluorescence microscopy. This report introduces procedures for the preparation of CLON-G and showcases an in vitro spontaneous death assay of neutrophils, which can be used for the study of neutrophils and for subsequently interrogating neutrophil death, thus providing a reliable resource for the neutrophil community.

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

This protocol details the preparation of CLON-G to extend the neutrophil lifespan to greater than 5 days and provides a reliable procedure for evaluating neutrophil death with flow cytometry and confocal fluorescence microscopy.

CLON-G 및 In Vitro Spontaneous Death Assay를 통한 호중구 수명 연장

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous ...