이 프로젝트의 자금은 국립 과학 재단 (National Science Foundation)을 부여 OCE1029607 (XM)을하고 MCB0702125 (MAM)을하고 고든과 베티 무어 재단 (MAM)을 제공한했습니다.

1. 워터 샘플 처리 <ol><li> 1 μm의 - 기공 크기의 멤브레인 필터를 통해 필터 10L 환경 물은 큰 입자와 bacteriovores을 제거합니다. 상자 속에 든 대형 유리병에 물을 여과물를 수집합니다. </li><li> 4 ML 신선한 paraformaldehyde 용액 (; 10% PFA)를 포함하는 3 멸균 Eppendorf 튜브 (50 ML)로 전송 36 ML 각 여과물. 세포를 보존하기 위해 상온에서 2 시간을위한 부화. 진공 여과에 의해 0.22 - μm의 - 기공 크기의 흰색 멤브레인 필터에 세포를 수집합니다. 진공 여과에 의한 필터를 통해 호수 (PBS)를 인산 버퍼 10 ML를 전달하여 필터를 씻으십시오. 부정적인 컨트롤로 필터를 라벨 및 -20 ° C.에 그들을 저장 </li></ol> 단계 1.3-1.4 DOC - 제한 조건을 설정을위한 옵션입니다. <ol start="3"><li> (5 μm의 NH 4 Club 호텔, 5 μm의 나노 3, 1μM 아니 2 PO 4 최종 농도) 상자 속에 든 대형 유리병에 무기 질소와 인의 혼합물을 추가합니다. </li><li> 가끔 agitations 48 시간 동안 현장의 온도에서 어둠 속에 품어. </li></ol> 2. 소우주 설립과 부화 <ol><li> microcosms을 설정하는 단계 1.4의 상자 속에 든 대형 유리병에서 800 ML 물 샘플 여섯 1 - L 유리 flasks 각각 입력합니다. 각각의 소우주로 BrdU (10 μm의 최종 농도)을 추가합니다. 잘 섞는다. </li><li> microcosms 세에 한 ML 모델 DOC 화합물 솔루션을 추가, 이들은 DOC의 개정안이 될 것입니다. 세 남아 microcosms로 한 ML 멸균 PBS를 추가, 이들은 노 또한 컨트롤 역할을합니다. </li><li> 보육 흔드는 모든 microcosms을 품어 100 rpm으로 흔들어하면서 현장의 온도에서 어둠 속에 품어. </li><li> 시간 0 점, 8, 16, 24 시간 50 ML 살균 Eppendorf 튜브에 각각의 축소판 및 전송에서 물 샘플 36 ML를 수집합니다. 즉시 수집 튜브에 신선한 PFA 4 ML (10 %)를 추가하고 세포를 보존하기 위해 상온에서 2 시간에 대해 숙고하다. </li><li> 필터는 0.22 - μm의 - 기공 크기의 폴리 카보 네이트 막 필터를 통해 세포를 보존. 10 ML PBS로 필터를 씻으십시오. 다음 단계로 바로 진행 또는 -20 ° C.에 필터를 저장 </li></ol> 3. BrdU의 설립에 대한해라 면역에 <ol><li> 상온에서 필터 (DOC 개정 샘플, 노 또한 컨트롤과 부정적인 컨트롤) 해동. </li><li> 라이 소 자임 솔루션 1 ML을 적용합니다 [10 100 MM 트리스, 50 NM EDTA (에틸렌 다이아 민 테트라 초산)에서 MG / ML 라이 소 자임 흰자위 (산도 = 8)] 필터에 박테리아 세포를 충당하기 위해 4. 30 분 실온에서 알을 품다. 흡입에 따라 그것을 통해 10 ML PBS를 전달하여 필터를 씻으십시오. </li><li> proteinase K 용액 1 ML 추가 [2 100 MM 트리스, 50 NM EDTA (에틸렌 다이아 민 테트라 초산)에서 MG / ML proteinase K (산도 = 8)] 필터에 박테리아 세포를 충당하기 위해 4. 30 분 실온에서 알을 품다. 흡입에 따라 그것을 통해 10 ML PBS를 전달하여 필터를 씻으십시오. </li><li> 1 ML의 exonuclease 솔루션 [exonuclease III (50 U / ML) 5 MM의 MgCl 2, 50 MM 트리스 - HCL] 5 필터에 박테리아 세포를 표지에 추가합니다. 37 품어 ° C를 30 분. 흡입에 따라 그것을 통해 10 ML PBS를 전달하여 필터를 씻으십시오. </li><li> 제조 업체의 지시에 따라 프레임 씰 배양 챔버 (생물 래드)를 조립. </li><li> 알코올 세척 건조 표면에 살균 블레이드를 사용하여 eighths에 필터를 슬라이스. </li><li> 포셉를 사용하여 하나의 조립 프레임 씰 배양 챔버 (8 프레임 인감 챔버 스가 각 필터 샘플이 필요합니다)에 필터의 여덟째 섹션을 놓으십시오. 세포를 포함하는 필터 부분의 측면 위쪽 얼굴 것입니다. 필터의 뒷면에있는 수분은 슬라이드 필터 막대기를하실 수 있습니다. 필터가 건조되면, 슬라이드에있는 필터 섹션을 배치하기 전에 챔버의 중앙에 diH 2 O의 작은 방울 (2 μl)를 적용할 수 있습니다. </li></ol> 단계 3.8-3.20는 제조 업체의 지침에서 변경 절차에 따라 현장 세포 증식 키트에서 호슈에서 시약을 FLUOS 사용합니다. PBS를 제외하고, 모든 시약은 키트 안에 제공됩니다. <ol start="8"><li> 균일하게 도장이 적용되면 오버플로가 발생하지 않고 배양 챔버의 전체 필터 부분을 커버하기에 충분한 배양 버퍼 (0.5 % 소 혈청 횐자위, PBS에 0.​​1 % Tween20) 적용됩니다. </li><li> 배양 챔버 프레임을 통해 폴리 에스테르 프레임 커버를 놓습니다. 단단히 배양 챔버를 밀봉 아래로 누르십시오. 필터 위에 기포를 사용하지 마십시오. 어둠 속에서 실온에서 10 분에 대한 봉쇄 실 품어. </li><li> 폴리 에스테르 도장을 제거하고 배양 챔버 스를 엽니다. (참고 : 챔버를 열고 때로는 슬라이드에서 프레임 인감 끌어 올려서 그 경우에는 다음 단계에 대한 새로운 챔버를 준비..) </li><li> 챔버 한 구석에서 부화 버퍼 아웃 Pipet. 필터와 접촉을 피하십시오. </li><li> 부드럽게 100 μl PBS를 pipetting하여 필터를 세척하고챔버에서 밖으로 3 회. </li><li> 바로 사용하기 전에 제조 업체에서 권장하는 단계를 다음과 같은 안티 - BrdU - FLUOS 작업 솔루션을 준비합니다. </li><li> Pipet 120 μl 방지 BrdU - FLUOS, 필터에 솔루션을 작업을 균일하게하여 전체 표면을 커버주의한다. </li><li> 새로운 폴리 에스테르 커버와 챔버를 봉인. 필터 위에 공기 방울을 피하십시오. 37 어둠의 챔버를 품어 ° C를 3 시간 동안. 이 단계는 플루오레신 isothiocyanate (FITC)와 현장에서 BrdU 통합의 DNA 라벨을 것입니다. </li><li> 폴리 에스테르 커버를 제거하고 배양 챔버를 엽니다. 안티 BrdU - FLUOS 작업 솔루션을 밖으로 Pipet. PBS와 필터 3 번 씻으십시오. </li><li> 배양 챔버에서 멸균 표면에 필터를 전송합니다. 멸균 블레이드를 사용하여 작은 조각으로 필터 섹션을 얇게. </li><li> 2 ML microcentrifuge 튜브, 1 ML PBS를 포함하는 각에 필터 조각을 전송합니다. 단단히 parafilm으로 튜브와 인감을 모자. 37 품어 ° C와 10 분, 200 RPM. </li><li> 5 분 최대 속도 vortexer과 소용돌이로 튜브를 고정합니다. 멸균 15 ML Eppendorf 튜브에 Pipet 뜨는합니다. 5 번 이상을위한 인큐베이션 및 와동의 단계를 반복합니다. 각 샘플에 대해 동일한 15 ML Eppendorf 튜브에 뜨는을 결합합니다. 일반적으로 80 % 세포 현탁액에서 복구할 수 있습니다. </li><li> 4 resuspended 세포와 표면에 뜨는를 저장 ° C. 2 일 이내에 정렬합니다. </li></ol> 4. 외과 분석 BD FACSAria 흐름 cytometer와 해당 소프트웨어에 대한 절차는 여기에 설명되어 있습니다. <ol><li> 제조 업체에서 권장하는 흐름 cytometer의 다음 단계의 설정을 최적화합니다. 이 알아볼 필요 breakoff 매개 변수와 달콤한 자리를 설정하는 증폭 제어를 조정하고, 실험 쉬스 압력 레이저 지연과 영역 확장 요소를 최적화하고 FSC와 SSC 전압, FSC 임계값, FSC 형광 스케일링, 형광 PMT 전압 설정을 최적화 등 </li><li> FITC 측면 분산형 (SSC)의 형광 강도에 따라 흐름 cytometer (FCM)에 부정적인 컨트롤 샘플을 실행합니다. 부정적인 컨트롤에 세포가 FITC - SSC 수집 디스플레이에 의​​해 시각을 사용할 수 없습니다 때까지 FITC 임계값을 높이십시오. </li><li> 노 또한 제어 샘플을 실행하고 FITC와 SSC 인수에 따라 만 세포의 분포 패턴을 확인합니다. 모든 세포를 싸주십시오 그리고 &quot;낮은 강도 전지&quot;(LIS)로 지정하기위한 게이트를 정의합니다. </li><li> DOC - 개정 샘플을 실행하고 FITC와 SSC에 따라 만 세포의 분포 패턴을 확인합니다. 일부 세포는 미리 LI 게이트에 나타납니다. LIS보다 높은 형광 강도를 (그의)가 세포를 묶으 다른 게이트를 정의합니다. 문이 세포의 상대적인 풍부를 볼 수 통계를 수집. </li><li> &quot;정화 한 방울&quot;모드에서 500 μl PBS를 포함하는 컬렉션을 튜브로 정렬 HI 세포. 그의 수가 500,000 카운트에 도달할 때 정렬을 종료할 수 있습니다. </li></ol> 5. 16의 필터 PCR 증폭은 rRNA 유전자를 필터 PCR 절차 Kirchman 외에서 수정 6. <ol><li> 흰색에 필터 정렬 세포, 0.22 μm의 - 기공 크기, 25mm 지름, 폴리 카보 네이트 멤브레인 필터. 멸균 블레이드를 사용하여 어떤 박테리아 세포를 포함하지 필터의 가장자리를 낸다. 8 동등한 크기의 조각으로 필터를 슬라이스. </li><li> 세포가 튜브의 안쪽을 향하고과 PCR 반응 관에 하나의 필터 부분을 넣으십시오. PCR 반응 관에 45 μl PCR 등급 물을 추가합니다. 물 속에 완전히 필터 잠수함. </li><li> 이 호슈 illustra PuReTaq 읽기 - 투 - 고, PCR 반응 관에 PCR 비즈를 간단히 와동을 추가합니다. PCR 반응 튜브의 각으로, 2 μl 같은 27F 및 1492R 7로 순방향 및 역방향 16 rRNA 유전자 primers (0.4 μm의 각 프라이머에 대한 최종 농도)를 각각 추가합니다. </li><li> (선택 사항) adsorb 증폭 억제제를 돕기 위해 PCR 반응 혼합물에 1μl 소 혈청 알부민 솔루션 (BSA, 최종 농도가 30μg/100 μl)을 추가합니다. 한 BSA를 추가하지 선택한 경우, 대신에 물을 1μl 추가할 수 있습니다. </li><li> 열 자전거 타는 사람에 PCR 증폭을 수행합니다. PCR 프로그램 다운 터치 권장합니다, 이것은 순차적으로 62에서 52로 감소 어닐링 온도를 가지고 ° C 1 ° C 52 어닐링 온도와 15주기에 의해 다음 11주기, ° C.에 대한 사이클당 모든주기는 (95 ° C에서) denaturing (62에서 52 ° C) 어닐링, 및 확장 (72 ° C에서) 50 기간의 단계를 포함합니다. 초기 3 분 변성 및 최종 10 분 확장 단계는 PCR 프로그램에 포함되어 있습니다. </li><li> ethidium 브로마이드 묻은 1퍼센트 아가로 오스 겔 전기 영동에 의한 PCR의 증폭을 확인합니다. 엑사이스 PCR의 겔에서 amplicons 및 QIAGEN QIAquick 겔 추출 키트와 함께 청소하십시오. </li><li> 각 샘플에 대한 두 개의 추가 PCR amplifications을 수행, 각 시간이 새 필터 섹션을 사용합니다. PCR 젤 purificati 후에 함께 동일한 샘플의 amplicons을 수영장. 정화 16 rDNA의 amplicons 지금 같은 클론 라이브러리 구축 및 시퀀싱과 같은 taxonomic 식별을 허용 분자 분석의 여러 준비가되어 있습니다. </li></ol> 6. 대표 결과 : 대표 결과는 예를 들어 putrescine - 굴욕적인 박테리아의 연구를 사용하여 설명되어 있습니다. 물 샘플은 조지아 해안 사이트에서 수집하여 위에서 설명한 절차에 따라 처리되었습니다. 외과 분석 putrescine 또한 높은 BrdU의 설립 속도 (그림 1)을 나타내는 높은 FITC 형광 강도와 박테리아의 그룹의 발전을 유도하는 것으로 나타났습니다. 이 전지는 높은 BrdU - 정관 전지 (그의)으로 지정되었으며 대부분 putrescine - 굴욕적인 박테리아를 포함하는 것으로 예상했다. HIS는 BrdU의 설립 (LIS)의 낮은 수준의 세포가 포함된 노 또한 컨트롤에서 누락되었습니다. LIS는 주로 추가 putrescine를 사용할 수 없습니다 bacterioplankton를 포함할 것으로 예상했다. 그의 분리 튜브에 정렬하고 멤브레인 필터로 수집했다. 16 rRNA 유전자 amplicons는 필터 PCR을 사용하여 높은 HI 세포에 대한 취득했다. <img src="/files/ftp_upload/2855/2855fig1.jpg" alt="그림 1"/> 그림 1. 노 또한 제어 및 부화 후 24 H 수집 (여기서 예로 putrescine) 모델 화합물 - 개정 표본의 흐름 cytometric 분석. 세포 분포 분석은 긍정적으로 BrdU의 정관의 수준과 관련이 FITC 라벨 (X 축) (1) 형광 강도에 따라 있었고, (2) 사이드 분산형 (SSC, Y 축)은, 긍정적으로 세포에 관련된 어떤 크기. 게이트 표기법은 BrdU의 설립, (; LI, 낮은 BrdU - 정관 HI, 높은 BrdU - 정관)의 수준에 따라 달라집니다. HI 및 LI 세포의 상대적 비율은 해당 게이트에 표시됩니다.

<ol>
	<li>Pernthaler, A., Pernthaler, J., Schattenhofer, M., Amann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+DNA-synthesizing+bacterial+cells+in+coastal+North+Sea+plankton.">Identification of DNA-synthesizing bacterial cells in coastal North Sea plankton.</a> Appl. Environ. Microbiol. 68, 5728-5728 (2002).</li><li>Urbach, E., Vergin, K. L., Giovannoni, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunochemical+detection+and+isolation+of+DNA+from+metabolically+active+bacteria.">Immunochemical detection and isolation of DNA from metabolically active bacteria.</a> Appl. Environ. Microbiol. 65, 1207-12 (1999).</li><li>Mou, X. Z., Hodson, R. E., Moran, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterioplankton+assemblages+transforming+dissolved+organic+compounds+in+coastal+seawater.">Bacterioplankton assemblages transforming dissolved organic compounds in coastal seawater.</a> Environ. Microbiol. 9, 2025-2025 (2007).</li><li>Hodson, R. E., Dustman, W. A., Garg, R. P., Moran, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+PCR+for+visualization+of+microscale+distribution+of+specific+genes+and+gene+products+in+prokaryotic+communities.">In situ PCR for visualization of microscale distribution of specific genes and gene products in prokaryotic communities.</a> Appl. Environ. Microbiol. 61, 4074-4074 (1995).</li><li>Dinjens, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bromodeoxyuridine+(BrdU)+immunocytochemistry+by+exonuclease+III+(Exo+III)+digestion.">Bromodeoxyuridine (BrdU) immunocytochemistry by exonuclease III (Exo III) digestion.</a> Histochemistry. 98, 199-199 (1992).</li><li>Kirchman, D. L., Yu, L. Y., Fuchs, B. M., Amann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+bacterial+communities+in+aquatic+systems+as+revealed+by+filter+PCR.">Structure of bacterial communities in aquatic systems as revealed by filter PCR.</a> Aquat. Microb. Ecol. 26, 13-13 (2001).</li><li>Delong, E. F., Wickham, G. S., Pace, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+stains:+ribosomal+RNA-based+probes+for+the+identification+of+single+cells.">Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells.</a> Science. 243, 1360-1360 (1989).</li><li>Artursson, V., Jansson, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+bromodeoxyuridine+immunocapture+to+identify+active+bacteria+associated+with+arbuscular+mycorrhizal+hyphae.">Use of bromodeoxyuridine immunocapture to identify active bacteria associated with arbuscular mycorrhizal hyphae.</a> Appl. Environ. Microbiol. 69, 6208-6208 (2003).</li><li>Mou, X. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+carbon+processing+by+generalist+species+in+the+coastal+ocean.">Bacterial carbon processing by generalist species in the coastal ocean.</a> Nature. 451, 708-708 (2008).</li><li>Mou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-cytometric+cell+sorting+and+subsequent+molecular+analyses+for+culture-independent+identification+of+bacterioplankton+involved+in+dimethylsulfoniopropionate+transformations.">Flow-cytometric cell sorting and subsequent molecular analyses for culture-independent identification of bacterioplankton involved in dimethylsulfoniopropionate transformations.</a> Appl. Environ. Microbiol. 71, 1405-1405 (2005).</li><li>Dean, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+human+genome+amplification+using+multiple+displacement+amplification.">Comprehensive human genome amplification using multiple displacement amplification.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 5261-5261 (2002).</li></ol>

관심 없음 충돌 선언하지 않습니다.

우리의 접근 커플 BrdU의 설립, 외과 및 16 rDNA 분석 수생 환경에서 개별 DOC 구성 요소를 대사 bacterioplankton의 종 수준의 식별을 허용합니다. BrdU의 설립 분석은 활성 세균에 대한 분석이 가능하기 때문에 휴면 세포를 포함하지 않습니다 신진 대사 활동을 기반으로 박테리아 세포를 표시합니다. 우리의 접근에서 BrdU의 설립은 이후 시각 아르 BrdU의 설립의 다른 수준을 가지고 immunodetected 현장과 박?...

<table border="1"><tbody><tr><td> 이름 </td><td> 유형 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> BrdU </td><td> 시약 </td><td> 시그마 </td><td> B5002 - 5G </td><td></td></tr><tr><td> 라이 소 자임 </td><td> 시약 </td><td> 시그마 </td><td> L6876 - 5G </td><td></td></tr><tr><td> Proteinase K </td><td> 시약 </td><td> 시그마 </td><td> P2308 - 25MG </td><td></td></tr><tr><td> 현장 세포 확산 키트, FLUOS에서 </td><td> 키트 </td><td> 로체 </td><td> 11810740001 </td><td> 안티 BrdU - FLUOS와 제조 업체가 제안보다 반응마다 부화 버퍼 더 소비합니다. </td></tr><tr><td> 프레임 시일 배양 챔버 </td><td> 자료 </td><td> 바이오 래드 </td><td> SLF - 1201 </td><td></td></tr><tr><td> 폴리 카보 네이트 멤브레인 필터 (142 - mm - 직경 1.0 μm의 - 구멍 크기) </td><td> 자료 </td><td> Millipore </td><td> FALP14250 </td><td></td></tr><tr><td> 폴리 카보 네이트 막 필터 (25 - mm - 직경 0.2 μm의 - 구멍 크기) </td><td> 자료 </td><td> Millipore </td><td> FGLP02500 </td><td></td></tr><tr><td> illustra PuReTaq 즉시 이동 PCR 비즈 </td><td> 키트 </td><td> GE 헬스케어 </td><td> 27-9559-01 </td><td></td></tr><tr><td> QIAquick 겔 추출 키트 </td><td> 키트 </td><td> QIAGEN </td><td> 28,704 </td><td></td></tr><tr><td> 안전 장치 ​​PCR 시스템 </td><td> 키트 </td><td> EPICENTRE </td><td> FS99060 </td><td></td></tr></tbody></table>

bromodeoxyuridine (brdu) labeling and subsequent fluorescence activated cell sorting for culture-independent identification of dissolved organic carbon-degrading bacterioplankton

미생물이 수중 환경에서 다수의 용해 유기 탄소 (DOC) 기판의 저하를 중재 주요 수단이된다. 그러나, 자연 DOC의 특정 풀을 변형 박테리아 taxa의 식별은 기술적인 도전을 포즈. 여기서 우리는 접근 방식을 설명하는 것을 커플 bromodeoxyuridine (BrdU) 정관, 형광 활성 세포 분류 (외과), 그리고 16 수생 환경에서 굴욕 특정 DOC 화합물 가능한 bacterioplankton 문화 독립적인 식별이 가능 rRNA 유전자 기반의 분자 분석. 세중의의 bacterioplankton의 microcosms은 BrdU와 모델 DOC 화합물 (DOC 개정) 또는 전용 BrdU (노 또한 컨트롤)를 모두 받도록 설정되었습니다. 새로 합성된 박테리아 DNA와 BrdU - 라벨 DNA의 thymidine의 BrdU의 대용품이 순위는 쉽게 immunodetected 1,2 수 있습니다. 추가 DOC 화합물을 선택적으로 활성화 될 것으로 예상하고, 따라서에서 노 DOC의 개정과 세포에 비 응답 전지보다 BrdU의 설립 높은 수준의 (HI 세포)를 가지고 있습니다 사용할 수 있습니다 24 시간 보육, bacterioplankton 통해 또한 컨트롤 (낮은 BrdU의 설립 세포, LI 전지). 형광 면역 후, HI 전지는 구별하고 육체적으로 형광 활성 세포 분류 (외과) 3 LI 세포 분리. 정렬 DOC - 반응 전지 (HI 세포)의 DNA가 추출 taxonomically PCR, 클론 라이브러리 구축 및 시퀀싱을 포함하여 rRNA 유전자 기반의 분석 이후 16를 통해 식별됩니다.

Watch this Scientific Journal Video about Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degradin

Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

환경 bacterioplankton는 유기 탄소 (DOC) 화합물과 DNA 라벨 시약, bromodeoxyuridine (BrdU)를 해산 모델 incubated 있습니다. 그 후, DOC - 굴욕적인 세포는 형광 활성 세포 분류 (외과)를 사용하여 고가 BrdU의 설립에 따라 대량 지​​역 사회에서 분리됩니다. 이러한 전지는 다음 이후의 분자 분석에 의해 식별됩니다.

Bromodeoxyuridine (BrdU) 레이블과 녹아 유기 문화 독립적인 확인을위한 후속 형광 활성 세포 정렬 탄소 굴욕 Bacterioplankton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification ...

bromodeoxyuridine-brdu-labeling-subsequent-fluorescence-activated

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Biology

Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

20.9K 조회수.  Kent State University. 환경 bacterioplankton는 유기 탄소 (DOC) 화합물과 DNA 라벨 시약, bromodeoxyuridine (BrdU)를 해산 모델 incubated 있습니다. 그 후, DOC - 굴욕적인 세포는 형광 활성 세포 분류 (외과)를 사용하여 고가 BrdU의 설립에 따라 대량 지​​역 사회에서 분리됩니다. 이러한 전지는 다음 이후의 분자 분석에 의해 식별됩니다.

비디오: Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

인간 CD40 - 활성화된 B 세포의 생성

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

연구

생물학

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

식물 Protoplasts의 형광 활성 세포 정렬

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

자기 활성 셀 정렬 (LIMACS)를 사용하여 지질 소포 - 중재 동질 크로마 토그래피 : 단백질 지질 상호 작용을 분석하는 소설 방법

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

의 세포 구체적인 분석<em&gt; Arabidopsis</em&gt; 형광 활성화 셀 정렬을 사용하여 잎

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, 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 ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

형광 활성 세포 정렬 (FACS)에 의해 신선한 조직으로부터 정상 및 암 관련 섬유 아세포의 분리

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Methyltransferases과 공동 인자 유사체와 핵산과 단백질의 순서 특정 레이블

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

마그네틱 활성화 전략을 분리 하 고 정화 활 액 액체 파생 된 중간 엽 줄기 세포는 토끼 모델에서 정렬 셀

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

거대한 바이러스 혼합물 분리를 위한 형광 활성화 세포 분류에 대한 대체 전략으로서의 단일 세포 마이크로 포부

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the mechanisms are not well understood. To investigate coral response to stressors, we need tools for analyzing cellular responses. In particular, we need tools that facilitate the application of functional assays to better understand how cell populations are reacting to stress. In the current study, we use fluorescence-activated cell sorting (FACS) to isolate and separate different cell populations in stony corals. This protocol includes: (1) the separation of coral tissues from the skeleton, (2) creation of a single cell suspension, (3) labeling the coral cells using various markers for flow cytometry, and (4) gating and cell sorting strategies. This method will enable researchers to work on corals at the cellular level for analysis, functional assays, and gene expression studies of different cell populations.

Corals create biodiverse ecosystems important for both humans and marine organisms. However, we still do not understand the full potential and function of many coral cells. Here, we present a protocol developed for the isolation, labeling, and separation of stony coral cell populations.

Scleractinian 세포 인구의 격리를 위한 형광 활성화 세포 분류

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the ...