Name: modified annexin v/propidium iodide apoptosis assay for accurate assessment of cell death
Uploaded: 2011-04-24T14:00:00.000+00:00
Duration: 7 min 34 s
Description: Watch this Scientific Journal Video about Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death at JoVE.com

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.

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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.

<table><tbody><tr><td>1 x PBS-/-</td><td></td><td></td><td></td></tr><tr><td>1 x Annexin V Binding Buffer</td><td>BD Biosciences</td><td>556454</td><td></td></tr><tr><td>Annexin V-Alexa Fluor 488</td><td>Molecular Probes, Life Technologies</td><td>A13201</td><td></td></tr><tr><td>Propidium iodide (PI)</td><td>Sigma-Aldrich</td><td>P4864</td><td>1 mg/mL in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>2% Formaldehyde</td><td>Sigma-Aldrich</td><td>F1268</td><td></td></tr><tr><td>RNase A from bovine pancreas</td><td>Sigma-Aldrich</td><td>R4642</td><td></td></tr><tr><td>DRAQ5</td><td>Biostatus</td><td>DR50050</td><td>Dilute 1:60 in 1 x PBS-/-</td></tr></tbody></table>

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 ...

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Research

JoVE Journal

Biology

103.1K 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

Detection and Isolation of Apoptotic Bodies to High Purity

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. It has been proposed that ApoBDs can aid cell clearance as well as intercellular communication through trafficking biomolecules. Conventional approaches used for the identification and isolation of ApoBDs are often limited by the lack of accurate quantification and low sample purity. Here, we describe a workflow to confirm the induction of apoptosis, validate ApoBD formation, and isolate ApoBDs to high purity. We will also outline and compare fluorescence-activated cell sorting (FACS) and differential centrifugation based approaches to isolate ApoBDs. Furthermore, the purity of isolated ApoBDs will be confirmed using a previously establish flow cytometry-based staining and analytical method. Taken together, using the described approach, THP-1 monocyte apoptosis and apoptotic cell disassembly was induced and validated, and ApoBD generated from THP-1 monocytes were isolated to a purity of 97-99%.

A workflow using flow cytometry or differential centrifugation is developed to detect, quantify and isolate apoptotic bodies from an apoptotic sample to high purity.

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. ...

연구

생화학

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

An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes more complex, the goal of targeting mitochondria using various compounds that inflict mitochondrial damage (so-called mitocans) is becoming quite popular. Unfortunately, many existing cytotoxicity assays, such as those based on tetrazolium salts or resazurin require functional mitochondrial enzymes for their performance. The damage inflicted by compounds that target mitochondria often compromises the accuracy of these assays. Here, we describe a modified protocol based on differential staining with two fluorescent dyes, one of which is cell-permeant (Hoechst 33342) and the other of which is not (propidium iodide). The difference in staining allows living and dead cells to be discriminated. The assay is amenable to automated microscopy and image analysis, which increases throughput and reduces bias. This also allows the assay to be used in high-throughput fashion using 96-well plates, making it a viable option for drug discovery efforts, particularly when the drugs in question have some level of mitotoxicity. Importantly, results obtained by Hoechst/PI staining assay show increased consistency, both with trypan blue exclusion results and between biological replicates when the assay is compared to other methods.

The protocol describes a rapid, high-throughput, reliable, inexpensive, and unbiased assay for efficiently determining cellular viability. This assay is particularly useful when cells&#39; mitochondria have been damaged, which interferes with other assays. The assay uses automated counting of cells stained with two nuclear dyes &#8211; Hoechst 33342 and propidium iodide.

미토칸 세포 독성의 정확한 측정을 위한 자동화된 차동 핵 염색 분석

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes ...

암 연구학

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 ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

신장 세포 Overexpressing 단백질 키나제 C Isozymes에 Mitochondrial 기능 및 세포 생존 능력의 평가

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

생물학

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

문화의 세포에 대한 생존 분석 실험

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

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 ...

An Introduction to Cell Death

Necrosis, apoptosis, and autophagic cell death are all manners in which cells can die, and these mechanisms can be induced by different stimuli, such as cell injury, low nutrient levels, or signaling proteins. Whereas necrosis is considered to be an &#8220;accidental&#8221; or unexpected form of cell death, evidence exists that apoptosis and autophagy are both programmed and &#8220;planned&#8221; by cells.In this introductory video, JoVE highlights key discoveries pertaining to cell death, including recent work done in worms that helped identify genes involved in apoptosis. We then explore questions asked by scientists studying cell death, some of which look at different death pathways and their interactions. Finally, several methods to assess cell death are discussed, and we note how researchers are applying these techniques in their experiments today.

Cell Death: Apoptosis, Autophagy and Necrosis

세포사에 대한 소개

Necrosis, apoptosis, and autophagic cell death are all manners in which cells can die, and these mechanisms can be induced by different stimuli, such as ...

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심화생물학

세포생물학

Annexin V and Propidium Iodide Labeling

Staining with annexin V and propidium iodide (PI) provides researchers with a way to identify different types of cell death&#8212;either necrosis or apoptosis. This technique relies on two components. The first, annexin V, is a protein that binds certain phospholipids called phosphatidylserines, which normally occur only in the inner, cytoplasm-facing leaflet of a cell&#8217;s membrane, but become &#8220;flipped&#8221; to the outer leaflet during the early stages of apoptosis. The second component is the DNA-binding dye molecule PI, which can only enter cells when their membranes are ruptured&#8212;a characteristic of both necrosis and late apoptosis.This video article begins with a review of the concepts behind annexin V and PI staining, and emphasizes how differential patterns of staining can be used to distinguish between cells progressing down different death pathways. We then review a generalized protocol for this technique, followed by a description of how researchers are currently using annexin V and PI staining to better understand cell death.

Cell Death Pathways and Annexin V &amp; PI Labeling studies

아넥신 V 및 프로피디움 아이오딘화물 라벨링

Staining with annexin V and propidium iodide (PI) provides researchers with a way to identify different types of cell death&#8212;either necrosis or ...