이 작품은 이글스 혁신 수상 (이글스의 성 오더)와 메이요 재단에 의해 지원되었다.

원시 단백질 - 단백질 상호 작용 및 유동세포계측법에 의해 multiprotein의 단지의 높은 감도 감지 및 정량 분석 : **이 비디오 프로토콜은 관련 출판 일을 기반으로합니다. 아담 G. Schrum, 다이애나 길, 일레인 P. Dopfer, 데이비드 L. 위스트, 로렌스 A. Turka, 볼프강 WA Schamel, 에드 파머. 과학의 STKE 2007 (389) : pl2 6 월 5, 2007, [간접 : 10.1126/stke.3892007pl2]. <a href="http://stke.sciencemag.org/cgi/content/abstract/2007/389/pl2">본 자료를 보시려면 여기를 클릭하십시오</a> . 시작하기 전에 다음 수성 주식 솔루션 준비 : <table><tbody><tr><td> MES 커플링 버퍼 : </td><td> 25 스토어 ° C </td></tr><tr><td> MES, 산도 6.0 </td><td> 50 MM </td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) </td><td> 1 ㎜ </td></tr><tr><td> 칭 / / 스토리지 (QBS) 버퍼를 차단 : </td><td> 4 스토어 ° C </td></tr><tr><td> BSA </td><td> 1퍼센트 </td></tr><tr><td> 나트륨 azide </td><td> 0.02 % </td></tr><tr><td> PBS, 산도 7.4 </td><td> 1X </td></tr><tr><td> FCM 버퍼 : </td><td> 4 스토어 ° C </td></tr><tr><td> 트리스, 산도 7.4 </td><td> 50 MM </td></tr><tr><td> NaCl </td><td> 100 MM </td></tr><tr><td> 나트륨 azide </td><td> 0.02 % </td></tr><tr><td> FBS </td><td> 5% </td></tr><tr><td> PBS, 산도 7.4 </td><td> 1X </td></tr></tbody></table> 사용하기 전에 바로 준비 : <table><tbody><tr><td> EDAC - MES : </td><td> MES 커플링 버퍼 50 MG / ML의 EDAC 분말을 디졸브 </td></tr><tr><td> Digitonin 솔루션 (2 % W / V) : </td><td> 95 가열하여 DH 2 O에서 digitonin 가루를 해산 ° C 5 분에 대한 다음 얼음에 냉각 </td></tr><tr><td> 용해 완충액 : </td><td></td></tr><tr><td> 트리스, 산도 7.4 </td><td> 50 MM </td></tr><tr><td> NaCl </td><td> 150 음 </td></tr><tr><td> Digitonin 솔루션 </td><td> 1퍼센트 </td></tr><tr><td> 프로 테아제 억제제 </td><td> 1X </td></tr><tr><td> 얼음 계속 </td><td></td></tr></tbody></table> 1. 비즈로 mAb의 커플링 <ol><li> PBS에 diluting와 hemacytometer로 계산하여 구입한 주식의 구슬의 농도를 결정합니다. 계산을위한 구슬의 1:10000 희석과 함께 시작합니다. </li><li> 피펫 18 1.5 - ML의 microcentrifuge 관으로 X 10 6 구슬. </li><li> 각 세척 후 25 3 분 2만그램 ° C에서 centrifuging, 0.5-1.0 ML MES 커플링 버퍼에있는 비즈에게 2-3 번 씻으십시오. </li><li> 50 μL MES 커플링 버퍼에있는 구슬을 Resuspend. </li><li> 신선한 EDAC - MES 20 μL를 추가하여 비즈의 카르 복실 그룹을 활성화합니다. </li><li> 25 ° C 수동으로 최대 pipetting 아래로 약 15 분 가볍게 섞는다. </li><li> 25 3 분 20,000그램에 centrifuging ° C를 각 씻고 나서, 0.5-1.0 ML PBS의 활성화 비즈에게 2-3 번 씻으십시오. </li><li> 50 μL PBS에서 활성 비즈를 Resuspend. </li><li> 활성 비드 솔루션 IP mAb (0.2-1.0 MG / ML 재고 농도에서) 50 μL를 추가합니다. </li><li> 진동 흔드는에 튜브를 삽입하여 25 ° C에서 3-4시간위한 섞는다. 튜브의 바닥에있는 구슬을 정착 방지하기 위해 충분히 흔들어. </li><li> 와시 mAb - 결합 구슬은 0.5-1.0 ML PBS 2 3 회, 각 씻어 후 25 3 분 2만그램에서 ° C centrifuging. </li><li> 100 μL QBS 버퍼에있는 구슬을 Resuspend. 이러한 4 ° C.에 저장할 수 있습니다 </li><li> 구슬을 중단하는 것은 물론, PBS에서 1:200-1:10,000을 희석하고 hemacytometer로 계산됩니다. 농도는 각 IP 또는 풀다운 실험에 사용되는 구슬의 개수를 정확하게 제어할 수 있도록, 각 예비 배치에 대한 측정해야합니다. </li></ol> 2. 포스트 핵 Lysate 준비와 IP 용해 방법 및 최적 용해 조건의 셀 유형과 검사되는 단백질 단백질 상호 작용에 따라 달라집니다. 프로토콜 번호가 존재하고 응용 프로그램을 수정할 수 있습니다. <ol><li> 이러한 lymphocytes, 또는 5 20 X 10 6 &#39;작은&#39;세포를 Lyse X 10 등 macrophages 또는 종양 세포와 같은 6 &#39;대형&#39;세포, 얼음에 20 분에 대해 1.5 ML microcentrifuge 관 100 μL 신선한 용해 버퍼 인치 필요에 따라 용해 볼륨을 스케일. </li><li> 핵 및 불용성 세포 파편을 제거하려면, 4 10 분 ° C.에 대한 2만g에서 lysate를 원심 분리기 뜨는 유지하고 펠렛을 삭제. </li><li> 섞어 부드럽게 pipetting 0.5 X 10 명확히 lysate로 5-2.5 X 10 5 mAb 결합 구슬을 추가합니다. 우리가 사용하는 최소 볼륨단일 IP를 수행 5 μL입니다. </li><li> 추운 방에서 하룻밤에 수직 회전 바퀴 4시간에 놓습니다. 정착의 구슬을 방지하기 위해 충분한 속도로 설정하십시오. 그것은 회전하는 동안 튜브의 바닥에 남아 낮은 볼륨의 IP를 액체에 대한 허용됩니다. </li></ol> 3. Fluorochrome - 복합 항체와 비드 - 캡처 단백질의 탐색 <ol><li> 4 2만그램에서 centrifuging ° C를 각 씻어 후 3 분, 0.2-1.0 ML 얼음처럼 차가운 FCM 버퍼의 IP 비즈 두 번 씻으십시오. </li><li> 굴뚝 - 바닥 접시 5 개 이상의 1.5 ML microcentrifuge 튜브 또는 우물의 각에 100 μL FCM 버퍼와 나누어지는 20 μL의 구슬을 Resuspend. </li><li> 샘플로 fluorochrome - 복합 mAbs를 추가합니다. FCM은 공급 업체의 지시에 따라 또는 경험적으로 결정 농도에 따라 얼룩 수행합니다. 출발점으로, 튜브 당 또는 잘 주식 항체 솔루션의 0.2-1 μL를 (0.2-1.0 MG / ML에서) 추가하고 얼음에서 40 분 품어. </li><li> 세차 4 2만그램에서 centrifuging, 1.0 ML 얼음처럼 차가운 FCM 버퍼의 1.5 ML 튜브에 두 번 구슬을 탐색할 · 각 세척 후 3 분 C. 부드럽게 구슬을 방해하지 않도록 돌봐 각 세척 후 표면에 뜨는를 제거합니다. 굴뚝 - 바닥 접시, 4에서 5 분 1,000그램에 centrifuging, 0.2 ML 얼음처럼 차가운 FCM 버퍼로 두 번 씻어 ° C를 각각 씻고 나서. 멀티채널 pipettor이 단계에 유용합니다. 굴뚝 - 바닥 플레이트의 비즈에서 뜨는 제거하려면, &#39;영화&#39;는 접시 한 ​​다음, 계속 거꾸로 반면, 우물의 가장자리로부터 drips을 얼룩. 각 우물에 잔류 볼륨은 구슬을 포함, 약 20 μL 것입니다. </li><li> 샘플 당 200 μL FCM 버퍼에있는 구슬을 Resuspend. 표시 외과 튜브로 전송. 샘플 지금 FCM에 대한 준비가되어 있습니다. </li></ol> 4. FCM 취득 이 프로토콜에 설명되어있는 CML 비즈는 직경 3-5 μm의, 정지 마우스 림프구의 약 절반 직경입니다. 따라서 수동으로 규모 등록 비드 이벤트의 인구에 대한 순서로 전달 스캐터 (FSC) A 이득과 사이드 스캐터 (SSC) 전압을 증가시킬 필요가있을 수도 있습니다. 개인 IP 구슬은 단일 단단히 클러스터된 인구를 형성한다. 설정 및 문은 구슬 doublets와 찌꺼기를 제외 조정해야합니다. <ol><li> 이전 실행 구슬 또는 구슬 기준을 일부 cytometers에 샘플 입구 주변 비말 억제 슬리브를 제거합니다. 이 입구의 선택을 취소 한 샘플에서 다른 이월되는 구슬을 방지하므로, 칼집 유체는 샘플 사이에 똑 수 있습니다. </li><li> 부정과 긍정적인 형광 극단 모두 로그 스케일 X - 축에 동시에 나타나도록 설정을 조정 레이블이 지정되지 않은 구슬, 형광의 부정적인 제어 및 레인보우 보정 입자 (RCPs), 형광의 긍정적인 컨트롤을 사용합니다. 이것은 실험 샘플의 형광도 규모에 될 수 있도록합니다. 그래서 FSC와 SSC 매개 변수뿐만 아니라, 게이트 위치가 일시적으로 해당 샘플에 대한 변경해야 할 수도, RCPs은 CML 비즈보다 작은됩니다. </li><li> RCPs과 교정이 완료되면 레이블이 지정되지 않은 - 구슬 설정으로 돌아갑니다. 샘플마다 하나의 형광 프로브를 사용하는 경우에는 형광 보정이 필요하지 않습니다. 일반적으로, 우리는 FCM 샘플마다 하나의 fluorochrome - 복합 mAb와 multiprotein의 단지를 탐사하고, 우리는 다양한 subunits 특정 mAbs과 병행하여 비드 샘플을 얼룩하여 여러 상호 작용 파트너를 확인합니다. </li><li> 형광 데이터 파일을 수집하고 저장합니다. 분석은 다음 유동세포계측법와 같은 CellQuest, FlowJo으로 소프트웨어 또는 CFlow를 사용하여 수행할 수 있습니다. </li></ol> 5. 대표 결과 <img src="/files/ftp_upload/2066/2066fig1.jpg" alt="그림 1" /> 그림 1. 마우스 변형에서 TCR/CD3 multiprotein 복합을위한 IP - FCM. T 세포는 BALB / C는 1 % digitonin에 lysed 있었고, lysate는 안티 - CD3ε 비즈를 사용하여 IP - FCM를 받게되었다. 캡쳐 단지는 TCR - β의 상당 수량 (보라색 지역)을 포함하고 공동 관련된 인 경우에는 3 번 CD - ε (녹색)하지만, 가까운 같은 Thy1.2 같은 다른 단백질의 배경 수준 (없는 면역 글로불린 프로브, 핑크 추적에 의해 정의)에, CD45, 또는 H - 2K (D) (갈색, 오렌지, 파랑 흔적, 각각). 비슷한 방식으로 이전에 게시된 결과 1-6을 참조하십시오.

<ol>
	<li>Schrum, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-sensitivity+detection+and+quantitative+analysis+of+native+protein-protein+interactions+and+multiprotein+complexes+by+flow+cytometry.">High-sensitivity detection and quantitative analysis of native protein-protein interactions and multiprotein complexes by flow cytometry.</a> Sci STKE. , pl2-pl2 (2007).</li><li>Schrum, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+multiprotein+complexes+by+flow+cytometry.">Visualization of multiprotein complexes by flow cytometry.</a> Curr Protoc Immunol. Chapter 5, 9-9 (2009).</li><li>Teixeiro, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+division+and+death+are+segregated+by+mutation+of+TCRbeta+chain+constant+domains.">T cell division and death are segregated by mutation of TCRbeta chain constant domains.</a> Immunity. 21, 515-526 (2004).</li><li>Teixeiro, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+T+Cell+receptor+signals+determine+CD8++memory+versus+effector+development.">Different T Cell receptor signals determine CD8+ memory versus effector development.</a> Science. 323, 502-505 (2009).</li><li>Treves, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Junctate+is+a+key+element+in+calcium+entry+induced+by+activation+of+InsP3+receptors+and/or+calcium+store+depletion.">Junctate is a key element in calcium entry induced by activation of InsP3 receptors and/or calcium store depletion.</a> J Cell Biol. 166, 537-548 (2004).</li><li>Gil, D., Schrum, A. G., Alarcon, B., Palmer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+engagement+by+peptide-MHC+ligands+induces+a+conformational+change+in+the+CD3+complex+of+thymocytes.">T cell receptor engagement by peptide-MHC ligands induces a conformational change in the CD3 complex of thymocytes.</a> J Exp Med. 201, 517-522 (2005).</li></ol>

단백질 - 단백질 상호 작용에 대한 정보는 신호 전달, 가계의 성숙, 세포주기 진행 및 apoptosis의 폭포 많은 세포 프로세스의 분석 관련성이있다. IP - FCM은 단백질의 상호 작용을 검토하고 원시 형태의 multiprotein의 단지의 멤버를 정의하기 위해, 빠른 양적 및 민감한 방법을 제공합니다. 비즈는 결합 및 세포 lysates incubated로 하루에와 탐지하고 그 다음날 분석할 수 있습니다. 96 - 웰 플레이트 형식은 통계 또는 검사 목적을 위해 효율적인 데이터 수집을 제공하기 위해 한 번에 분석하기 위해 샘플을 다수의 허용합니다. 형광 비드 표준을 사용하여 각 비드 점령 단백질의 수를 추정 수 있습니다. 거의 원본 자료는 캡처 및 탐지에 필요한이므로 제한된 샘플 및 부족 analytes은 여전히​​ 여러 상호 작용에 대한 분석을하실 수 있습니다. IP - FCM이 아닌 생리적 환경에서 단백질의 혼합 유전 공학, 에피토프 - 태그, 변성, 또는 - 체외 필요하지 않지만, 그것은 그것에 적용과 가치 및 접근 도구를 만들고, 이들과 다른 기술을 결합 수 있습니다 많은 생물 학적 시스템. 문제 해결 : 대부분의 IP - FCM 실험에 유용 단백질 상호 작용 데이터에게 그들이 시도하는 처음 생성합니다. 그러나, IP - FCM의 최적화 구슬, 단백질 복잡한 캡처 및 바인딩 형광 프로브에 항체 활용을 향상시킬 수 있습니다. IP 항체 활용의 효율을 방지 면역 글로불린 항체와 직접 결합 구슬을 탐색하여 결정하실 수 있습니다. 이 효율이 낮은 경우, 커플링 반응 중에 항체의 농도를 증가하는 것은 많은 IP 항체가 각각의 구슬에 첨부할 수 있습니다. 이것은 analytes의 향상된 캡처 및 검색의 결과로, IP 비드 배치의 바인딩 용량을 늘릴 수 있습니다. 이러한 낮은 mAb 커플링의 비드 부착 및 결과에 대한 mAb와 경쟁할 수와 같은 다른 기본 - 아민 포함하는 분자 (예 : 트리스, 소 혈청 알부민), 커플링 반응 동안에 존재해서는 안됩니다. 구슬로 mAb의 활용이 다른 이유로 문제가 증명하는 경우, 구슬 대신 아비딘 / streptavidin을 결합 수 있으며, biotinylated 항체는 이후 비 covalently 바운드 및 immunprecipitation 사용할 수 있습니다. 좋은 항체 활용에도 불구하고, 비드 - 관련 형광의 초기 감지는 낮은 수 있습니다. 첫째, 복잡한 촬영 자체가 적을 수 있습니다. IP - FCM은 단위 용해 량 당 lysed 세포의 수를 증가 analytes의 농도에 의존하기 때문에 analyte 캡처 및 감지를 높일 수 있습니다. 또한 캡처는 캡처 이하의 구슬에 걸쳐 단지, 그리고 탐지 비드 당 평균 형광 증가의 결과를 배포 lysate와 함께 incubated IP 구슬의 개수를 줄임으로써 향상된 수 있습니다. 둘째, 그것은 캡처한 단지에 프로브 항체의 액세스가 sterically immunoprecipitating 항체에 의해 방해되어 있습니다. 이 경우에는 문제가 및 / 또는 탐침을 캡처하는 다른 항체를 사용하여 해결할 수 있습니다.

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<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>carboxylate-modified polystyrene latex (CML) beads (5mm surfactant-free)</td>
<td>Reagent</td>
<td>Interfacial Dynamics Corporation/Molecular Probes</td>
<td>2-5000</td>
<td>Store at 4&deg;C.</td>
</tr>
<tr>
<td>Rainbow Calibration Particles</td>
<td>Reagent</td>
<td>Spherotech, Inc.</td>
<td>RCP-30-5A</td>
<td>Store at 4&deg;C.</td>
</tr>
<tr>
<td>2-[N-Morpholino] ethanesulfonic acid (MES)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M-5287</td>
<td>&nbsp;</td>
<tr>
<td>1-Ethyl-3-(3-dimethylaminopropl) carbodiimide HCl (EDAC)</td>
<td>Reagent</td>
<td>Pierce Sigma</td>
<td>22980 E-6383</td>
<td>Store at -20&deg;C under desiccating conditions.</td>
</tr>
<tr>
<td>Protease Inhibitor General Use Cocktail, containing AEBSF, Bestatin, Aprotinin, EDTA, E-64, and Leupeptin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P-2714</td>
<td> Prepare 100x stock solution in H2O. Aliquot and store at -20&deg;C. Handle with gloves.</td>
</tr>
<tr>
<td>PCR plates, 96-well 0.2mL, natural, tall chimney</td>
<td>Reagent</td>
<td>Fisher</td>
<td>08-408-230</td>
<td>For use in place of microcentrifuge tubes during FCM staining</td>
</tr>		</tbody>
		</table>
		</div>

ip-fcm:  immunoprecipitation detected by flow cytometry

유동세포계측법 (IP - FCM)에 의해 감지 Immunoprecipitation은 단백질 - 단백질 상호 작용을 감지하고 quantifying위한 효율적인 방법입니다. 기본 원리는 촬영한 기본 analyte가 실제로 multiprotein의 단지 내에 관련된 다른 분자와 함께 감지할 수했었습니다 샌드위치 엘리사의 그것을 확장합니다. 절차는 관심의 단백질에 대한 단클론 항체 (mAb) 특정을 immunoprecipitating 세포 lysates 이러한 구슬을 잠복기, fluorochrome - 복합 프로브와 함께 캡처 단백질 단지를 프로빙 및 유동세포계측법의 구슬 - 관련 형광을 분석과 폴리스티렌 라텍스 microbeads의 공유 결합 결합을 포함한다. IP - FCM은 매우 민감한 것입니다 자신의 모국어 (비 변성) 상태에서 단백질의 분석이 가능하고, 중 반 정량 또는 정량 분석​​ 의무가 있습니다. 추가 장점으로 IP - FCM은 흐름 cytometer 이외의 유전 공학이나 전문 장비를 필요로하지 않으며 그것은 쉽게 높은 처리량 어플 리케이션에 적용할 수 있습니다.

Watch this Scientific Journal Video about IP-FCM:  Immunoprecipitation Detected by Flow Cytometry at JoVE.com

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

유전 공학 또는 큰 샘플 크기를 요구하지 않고, 고유의 단백질 - 단백질 상호 작용의 구분, 강력하고, 생화학 평가를 허용하는 IP - FCM 방식은 제공됩니다.

IP - FCM : 유동세포계측법에 의해 감지 Immunoprecipitation

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

ip-fcm-immunoprecipitation-detected-by-flow-cytometry

Research

JoVE Journal

Biology

17.4K 조회수.  Mayo Clinic. 유전 공학 또는 큰 샘플 크기를 요구하지 않고, 고유의 단백질 - 단백질 상호 작용의 구분, 강력하고, 생화학 평가를 허용하는 IP - FCM 방식은 제공됩니다.

비디오: IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

유동세포계측법에 의해 플라즈마 막에 GLUT4 Translocation의 양적 측정

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

연구

생물학

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

마우스 Meiotic 셀 유동세포계측법의 정제

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

셀 추적 염료를 사용하여 전지 부문 모니터링에 최적화 된 착색 및 확산 모델링 방법

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

유동 세포 계측법에 의한 신장 상피 세포에서 세포 내 칼슘 측정

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

고립 된 미토콘드리아의 유동 세포 계측법에 의해 외부 막 단백질의 면역

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

유동 세포 계측법에 의한 고효율 심근에 인간 다 능성 줄기 세포의 분화 및 특성

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

LED 열 흐름 - 유동 세포 계측법으로 Optogenetics 결합

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

스펙트럼 유동 세포 계측법에 의해 고체 조직에서 분리 한 세포 현탁액의 분석

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

격리 된 괴 경 Protoplasts의 Cytometry에 의해 측정 Endoreduplication

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

Flow Cytometry 여 Dictyostelium discoideum Macropinocytosis의 높은 처리량 측정

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...