이 작품은 NIH 국립 심장 폐 및 혈액 연구소 부여 RO1 HL81248에 의해 재정 지원되었다.

1. 소의 대동맥 내피 세포 (BAEC)의 문화 <ol><li> 적절한 무균 기법을 미행했다. </li><li> 37 항생제없이 물 목욕, 따뜻한 매체 ° C로 </li></ol> 주 : 중간 4.5 G / L D-글루코오스와 페놀 무료 Dulbecco의 수정된 이글 배지 (DMEM)로 구성되어 10 % 태아 소 혈청 (FBS) 및 2.5과 함께 보완 4 MM L-글루타민, 1 %가 아닌 필수 아미노산, MG / L 내피 성장 인자. <ol start="3"><li> 인큐베이터에서 T75 플라스크 들어있는 세포를 제거하고 후드 안쪽에 그것을 배치하기 전에 70 %의 에탄올과 플라스크의 표면을 청소하십시오. </li><li> 흡인기를 사용하여 기존의 매체를 제거하고 Dulbecco의 인산염 완충 식염수 (DPBS) 5 ML로 두 번 씻는다. </li><li> 트립신 2 ML을 추가하고 정기적으로 현미경으로 검사하는 동안 분리 전지 4-5 분 기다립니다. </li><li> 매체 3 ML을 추가하고 반복적으로 별도의 t에 피펫을 사용하여 혼합그는 세포도 정지를 만듭니다. </li><li> 5 분 대한 121g에서 약 15 ML 튜브와 원심 분리기에 트립신과 매체의 5 ML을 전송합니다. </li><li> 흡인기를 사용하여 표면에 뜨는을 제거합니다. DPBS 5 ML을 추가하고 철저하게 섞는다. 5 분 동안 121g에서 원심 분리기. </li><li> 뜨는을 대기음 및 매체 6 ML을 추가하여 세포 펠렛을 다시 일시 중지합니다. </li><li> 6 - 잘 접시에, 그럼 중간 1 ML을 추가할 각 세포 현탁액 1 ML을 추가합니다. 피펫을 사용하여 정지를 섞습니다. </li><li> 접시 라벨과 37 하룻밤 품어 ° C에서 5 % CO 2. </li></ol> 2A. BAEC 전지와 NO의 탐지 <ol><li> 적절한 무균 기법을 미행했다. </li><li> 배양기에서 6 잘 플레이트를 제거하고 첫 번째 우물에서 매체를 기음. 한 ML의 DPBS으로 두 세포를 씻으십시오. </li><li> 1.9 MM 철 (II) 황산의 heptahydrate (FeSO 4 0.7 H 2 O에서 0.8 밀리그램을 분해하여 신선하게 준비 중 210 μl를 추가하십시오1시 7분의 비율을 사용하여 한 CaCl 2, MgCl 2 ML DPBS)와 암모늄 N-메틸-D-glucamine dithiocarbamate (MGD, CaCl 2, MgCl 2 500 μl DPBS에서 2.7 밀리그램을 분해하여 신선하게 준비)의 210 μl . (참고 :. 초과 MGD가 사용되는이 비율은 통상 철 3에 대한 수율은 + MGD이 초과 MGD의 면전에서 최대있다는 사실로 인해 + MGD 복잡한 철 2의 준비를 위해 고용되었습니다의 추가 철 2를 안정화하기위한 솔루션 아스코 브 산은 + MGD NO-철 3 이래로 형성된 + MGD는 endogenously 아스코 브 산, 히드로퀴논, 또는 최대 변환 효율과 시스테인으로 + MGD이 NO-철 EPR 감지로 줄어 듭니다 필요하지 않습니다 99.9 %로 .. 철 2의 낮은 스핀 diamagnetic 상태 + NO) 저온 장치의 필요없이 평면 셀 또는 모세관을 사용하여 15 검출이 가능합니다. </li><li> 소용돌이 결과 현탁액 잘하고 4.6 μ 추가칼슘 ionophore의 리터 (CAI) (1 ML DMSO에 1 밀리그램을 해소하여 1.9 mm의 주식 솔루션에서 준비). </li><li> 소용돌이 다시 솔루션 및 순서 36 분 대한 ° C에서 추가 - 철 NO 3의 감소는 NO-철 2 +-MGD EPR 감지에 + MGD 수 있도록 37 알을 품다. </li><li> Eppendorf 튜브 및 EPR 평평한 세포 (또는 50 μl 모세관까지) 이전에 뜨는 (425 μl)를 수집합니다. </li><li> EPR 수집 매개 변수는 다음과 같습니다 마이크 로파 주파수 : 9.8 GHz의, 중심 분야 : 3,427 G, 변조 진폭 : 6.0 G, 스위프 폭 : 100 G, 리시버 게인 : 1 × 10 5; 마이크 로파 전력 : 10 MW, 스캔의 총 개수 : 121; 스위프 시간 : 10의, 그리고 시정수 : 20 MS (참고 : 매개 변수가 하나의 악기와 실험 조건에서 다른 달라질 수 있기 때문에, 따라서만을 중심 분야, 주파수 및 변조 진폭은 고려해야 할 가장 중요한 매개 변수입니다.). </li><li> 실온에서 스펙트럼을 기록하며 2 차원 스펙트럼은 integ되었습니다Bruker WinEPR 데이터 처리 소프트웨어 또는 다른 데이터 처리 소프트웨어를 사용하여 수정 배경 소음 기준을 줄이기 위해 평가. adduct 형성 부량 들어, 집중력 대 신호 강도 (또는 지역)의 표준 줄거리는 기증자 스냅인 아니오를 사용하지 구축하실 수 있습니다. </li></ol> 2B. eNOS Uncoupling 실험 <ol><li> 배양기에서 6 잘 플레이트를 제거하고 두 번째 우물에서 매체를 대기음 1 ML의 DPBS로 두 번 씻는다. </li><li> peroxynitrite 기증자, 5 - 아미노 -3 - (4-morpholinyl) -1,2,3-oxadiazolium 염화물 (SIN-1)은 eNOS 16 개를 풀다하는 데 사용되었다. 0.5 밀리미터 SIN-1 (M R 206.6 갓 노 CA / MG 이온과 PBS 500 μl에 1 MG SIN-1을 해소하여 준비 10 밀리미터 주식 솔루션에서 G / 몰) 및 묽은 ~ 2 ML과 DPBS 100 μl를 추가하십시오 와 10 % FBS. </li><li> 37 2 시인데 동안 품어 ° C에서 5 % CO 2. </li><li> 인큐베이터에서 접시를 제거하고 DPBS로 두 번 씻는다. </li><li> 의 210 μl 추가2.8 MM FeSO 4 0.7 H 2 O와 19.6 mm의 210 μl와 신선한 위에서 언급한 절차에 따라 준비 MGD. </li><li> 소용돌이 솔루션 및 1.9 밀리미터 CAI의 4.6 μl를 추가합니다. </li><li> 소용돌이 다시 솔루션 및 36 분 동안 37 ° C에서 품어. </li><li> Eppendorf 튜브 및 EPR 평평한 감방으로 전송에서 (425 μl) 뜨는 수집합니다. </li><li> EPR 획득 파라미터는 다음과 같습니다 마이크로 웨이브 주파수 : 9.8 GHz의, 중심 분야 : 3,427 G, 변조 진폭 : 6 G, 스위프 폭 : 100 G, 리시버 게인 : 1 × 10 5; 마이크 로파 전력 : MW 10, 스캔의 총 개수 : 121; 스위프 시간 : 10의, 그리고 지속 시간 : 20 MS. </li><li> 스펙트럼을 기록하며 2 차원 스펙트럼은 위에서 언급한 수정 배경 소음 기준을 줄이기 위해 통합되었다. </li></ol> 3. O 2의 탐지 • - Polymorphonuclear Neutrophils에서 (PMNs) <ol><li> 이전 17 설명된대로 Neutrophils은 인간의 혈액 샘플에서 고립되었다. </li><li> diethylenetriamine-pentaacetic 아세트산 (DTPA) 0.1 밀리미터를 포함하는 PBS에 1 M DMPO *의 주식 솔루션을 확인하십시오. DMPO는 25-29의 융점을 가지고 ° C ~ 그것이 유리관 (주에 액체 DMPO을 (고밀도 ~ 1.02 G / ML 25 ° C) 피펫 것이 더 편리하므로 : 순수 DMPO 이후 무게를 위해 플라스틱 튜브를 사용하지 않습니다 ) 플라스틱과 반응. 냉동 DMPO는 유리병 (DMPO가 분해 수 있으므로 온수를 실행하지 않는 주)에 루크의 온수를 실행하여 녹아 수 있습니다. </li><li> 그것은 이후 고순도 DMPO를 (&gt; 99 %)를 사용하는 것이 중요 상용 스핀 트랩 중 일부는 상자 성체의 불순물을 포함하고, 따라서 그것만으로도 DMPO 솔루션의 EPR 스펙트럼 (이 경우 10 MM)를 실행하는 것이 필수적입니다. 그것은 배경 신호 (그림 3A 참조) 분명없는 것이 중요합니다. </li><li> DMSO의 phorbol-12-myristate-13-아세테이트 (PMA)의 주식 솔루션 (1 밀리그램 / ML) 준비합니다. 10 μg / PBS의 ML에 대한 솔루션을 diluting하여 aliquots하십시오. </li><li> 1.5 ML Eppendorf 튜브는 또한 순서에 따라 0.6 ML의 총 부피와 솔루션을 준비 : PMN의 ML 당 ~ 10 6 세포, D-글루코오스 (1 밀리그램 / ML) 및 알부민 (1 밀리그램 / ML), 10 MM DMPO와 PMA의 0.2 μg / ML. (참고 : PMA는 급진 활성제이며, 마지막으로 추가됩니다.) </li><li> EPR 편평한 세포에 대한 솔루션을 전송합니다. </li><li> EPR 인수 매개 변수 조건은 다음과 같습니다 마이크로 웨이브 주파수 : 9.8 GHz의, 중심 분야 : 3,486 G, 변조 진폭 : 0.5 G, 스위프 폭 : 100 G, 리시버 게인 : 5 × 10 5; 스캔의 총 개수 : 10, 스위프 시간 : 30의 ; 마이크 로파 전력 : 20 MW, 그리고 지속 시간 : 81 MS. adduct 형성 부량 들어, 집중력 대 신호 강도 (또는 지역)의 표준 줄거리는 같은 템포 또는 3 - 카르복실산-PROXYL 등 안정 nitroxides를 사용하여 건설 수 있습니다. </li></ol> DMPO의 사용에 대한 * 중요 참고 : 평면 세포를 사용함으로써, 하나는 세포 밀도를 높이고이를 통해 신호를 증가 수스핀 adduct지만, O 2의 반감기의 강도 • - DMPO의 adduct가 DMPO-OH로 분해하는 (T 2분의 1 ~ 1 분) 짧습니다. DMPO는 t 2분의 1 ~ 8, 각각 14 분, 함께 증가 adduct의 안정성을 위해 상업적으로 사용할 수 있습니다 EMPO, BMPO 또는 DEPMPO의 동일한 농도를 사용하여 대체 할 수 있습니다. 4. 대표 결과 스핀 NO 라디칼의 포집이 철 2 +-MGD. 그림 2A와 2B를 사용하여 수행되었다 철 2에서 더 EPR 신호를 보여주지 + MGD 또는 철 2의 혼합 아무런 배경 신호 어떠한 이러한 시약 유래 없다는 것을 나타내는 CAI와 + MGD.에게 CAI와 자극시 BAEC는 철 2 반응하는 아니오를 출시하지 스핀 adduct, NO-철 2 +-MGD을 형성 + MGD, 그리고 초미세 분리 상수 (hfsc) N = 12.66 G의 가치와 특성 삼인승 신호를 보여줍니다 g = 2.040의 G-요소. (그림 2C). HFSC 값이 NIEHS EPR 소프트웨어 데이터베이스 웹 사이트에서 다운로드할 수 있습니다 WINSIM 시뮬레이션 프로그램을 사용하여 결정되었다. 실험 hfsc은 NO-철 2 +-MGD adduct를위한 N = 12.70 G와 g = 2.041 18 문학의 가치와 일치합니다. 마찬가지로, BAEC에 SIN-1의 효과는 그림 2D와 같이 uncoupling eNOS에 의해 생산 아니오를 낮춘 없습니다. 감지 - DMPO 스핀 트랩이 O 2 • 위해 사용되었다. 스핀 트랩은 상자 성체의 불순물에서 무료임을 확인하는 그림 3A와 같이 혼자 DMPO은 신호를주지 않았다. 그림 3B는 DMPO의 스펙트럼과 PMNs에만, 그리고 마찬가지로, DMPO이 발생하지 않는다는 것을 제안 전혀 감지 신호가 없습니다 효소 NADPH 산화 효소의 활성화. 그림 3C는 PMA에 의한 PMN의 자극시 관찰된 EPR 신호를 보여줍니다. 이 신호에 대한 hfsc 값은 N = 14.71 G, &amp; 것으로 확인되었습니다베타,-H = 11.40 G ​​및 γ-H = 1.25 G, 그리고 N = 14.3 G, β-H = 11.7 G 및 γ-H의 문헌 값 DMPO-O 용 = 1.3 G 19 일치 2 시인데의 adduct. <img src="/files/ftp_upload/2810/2810fig1.jpg" alt="그림 1" /> 그림 1. EPR 스핀 트래핑을 사용하여 neutrophils과 BAEC에서 래디 칼의 검출을위한 플로우 차트. () PMNs는 DMPO와 PMA 섞인 있었고, 결과 혼합물은 데이터 수집을위한 EPR 평평한 세포로 전학 왔어. (B) BAEC는 접시에 성장하고, DPBS로 씻어되었습니다. 스핀 트랩 철 (MGD는) 2 CAI와 함께 추가되었습니다. 솔루션은 철저하게 혼합하고 incubated되었다. 혼합물은 EPR 데이터 수집을위한 EPR 평평한 세포로 이송되었다. <img src="/files/ftp_upload/2810/2810fig2.jpg" alt="그림 2" /> B에서 NO의 그림 2. EPR 검출AEC. 철 2 (A) 스펙트럼 + MGD 전용 (B) + MGD + CAI 전용 철 2의 스펙트럼. (C) 철 2로 NO 트래핑으로 인한 삼인승 스펙트럼이 + MGD 사용하여 세포를 CAI 것은 자극. (D)으로 인해 0.5 MM 세포의 SIN-1 처리에 어떠한 생산의 감소를 보여주는 스펙트럼. <img src="/files/ftp_upload/2810/2810fig3.jpg" alt="그림 3" /> 활성화 neutrophils에서 DMPO, O 2 H의 그림 3. EPR 검출. 10 밀리미터 DMPO 전용의 () 스펙트럼. (B) 10 밀리미터 DMPO의 앞에서 혼자 PMN의 스펙트럼. (C) PMN의 스펙트럼은 10 밀리미터 DMPO의 면전에서 PMA에 의해 활성화.

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U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decomposition+mechanism+of+3-N-morpholinosydnonimine+(SIN-1):+A+density+functional+study+on+intrinsic+structures+and+reactivities.">Decomposition mechanism of 3-N-morpholinosydnonimine (SIN-1): A density functional study on intrinsic structures and reactivities.</a> J. Mol. Model. 10, 121-129 (2004).</li><li>Klempner, M. S., Gallin, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+and+functional+characterization+of+human+neutrophil+subpopulations.">Separation and functional characterization of human neutrophil subpopulations.</a> Blood. 51, 659-669 (1978).</li><li>Pou, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spin+trapping+of+nitric+oxide+by+ferro-chelates:+kinetic+and+in+vivo+pharmacokinetic+studies.">Spin trapping of nitric oxide by ferro-chelates: kinetic and in vivo pharmacokinetic studies.</a> Biochim. Biophys. Acta. 1427, 216-226 (1999).</li><li>Finkelstein, E., Rosen, G. M., Rauckman, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spin+trapping.+Kinetics+of+the+reaction+of+superoxide+and+hydroxyl+radicals+with+nitrones.">Spin trapping. Kinetics of the reaction of superoxide and hydroxyl radicals with nitrones.</a> J. Am. Chem. Soc. 102, 4994-4999 (1980).</li><li>Britigan, B. E., Rosen, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spin-trapping+and+human+neutrophils.+Limits+of+detection+of+hydroxyl+radical.">Spin-trapping and human neutrophils. Limits of detection of hydroxyl radical.</a> J. Biol. Chem. 264, 12299-12302 (1989).</li><li>Frejaville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline+N-oxide:+A+new+efficient+phosphorylated+nitrone+for+the+in+vitro+and+in+vivo+spin+trapping+of+oxygen-centered+radicals.">5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide: A new efficient phosphorylated nitrone for the in vitro and in vivo spin trapping of oxygen-centered radicals.</a> J. Med. Chem. 38, 258-265 (1995).</li><li>Snyrychova, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+the+sensitivity+of+EPR+spin+trapping+in+biological+systems+by+cyclodextrins:+A+model+study+with+thylakoids+and+photosystem+II+particles.">Improvement of the sensitivity of EPR spin trapping in biological systems by cyclodextrins: A model study with thylakoids and photosystem II particles.</a> Free Radical Biol. Med. 48, 264-274 (2010).</li><li>Han, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipophilic+beta-cyclodextrin+cyclic-nitrone+conjugate:+Synthesis+and+spin+trapping+studies.">Lipophilic beta-cyclodextrin cyclic-nitrone conjugate: Synthesis and spin trapping studies.</a> J. Org. Chem. 74, 5369-5380 (2009).</li><li>Han, Y., Tuccio, B., Lauricella, R., Villamena, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+spin+trapping+properties+by+beta-cyclodextrin-cyclic+nitrone+conjugate.">Improved spin trapping properties by beta-cyclodextrin-cyclic nitrone conjugate.</a> J. Org. Chem. 73, 7108-7117 (2008).</li><li>Hardy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection,+characterization,+and+decay+kinetics+of+ROS+and+thiyl+adducts+of+mito-DEPMPO+spin+trap.">Detection, characterization, and decay kinetics of ROS and thiyl adducts of mito-DEPMPO spin trap.</a> Chem. Res. Toxicol. 20, 1053-1060 (2007).</li><li>Kim, S. -. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+reactivity+of+a+cyclic+nitrone-calix[4]pyrrole+conjugate+with+superoxide+radical+anion:+Theoretical+and+experimental+studies.">Fast reactivity of a cyclic nitrone-calix[4]pyrrole conjugate with superoxide radical anion: Theoretical and experimental studies.</a> J. Am. Chem. Soc. 132, 17157-17173 (2010).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

EPR의 스핀 트래핑은 자유 래디 칼을 quantifying 및 식별을 위해 생명 의학 애플 리케이션의 광범위한 채용되었습니다. 스핀 트래핑은 고도로 민감한 NM에서 따라서 생물 학적 시스템의 응용에 적합하게 μm의에 이르기까지 다양한 농도로 래디 칼을 감지 할 수있다. 상자 성체의의 adduct, NO-철 2 +-MGD의 형성은 EPR 통해 NO 검출의 기초입니다. ~의 속도로 10 6 M -1 s의 -1에?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 (옵션) </td></tr><tr><td> 페놀 무료 DMEM 배지 높은 포도당 1X </td><td> GIBCO </td><td> 31,053 </td><td></td></tr><tr><td> 0.25 % 트립신-EDTA (에틸렌 다이아 민 테트라 초산) </td><td> GIBCO </td><td> 25,200 </td><td></td></tr><tr><td> L-글루타민 </td><td> 피셔 과학 </td><td> BP379-100 </td><td></td></tr><tr><td> MEM 비 필수 아미노산 </td><td> GIBCO </td><td> 11,140 </td><td></td></tr><tr><td> 태아 소 혈청 </td><td> 애틀랜타 체액 </td><td> S11550 </td><td></td></tr><tr><td> 내피 성장 인자 </td><td> Millipore </td><td> 02-102 </td><td></td></tr><tr><td> CAI </td><td> 엔조 생명 과학 </td><td> A-23187 </td><td> DMSO에 용해 </td></tr><tr><td> SIN-1 </td><td> 엔조 생명 과학 </td><td> BML-CN245-0020 </td><td></td></tr><tr><td> DMPO </td><td> Dojindo 연구소 </td><td> D048-10 </td><td></td></tr><tr><td> FeSO 4 0.7 H 2 O </td><td> 시그마 알드리치 </td><td> 215422-250G </td><td> 칼슘과 MG와 PBS에 디졸브 </td></tr><tr><td> MGD </td><td> 엔조 생명 과학 </td><td> ALX-400-014 - M050 </td><td> 칼슘 2 + 및 MG 2 PBS에 디졸브 + </td></tr><tr><td> BAEC 세포 </td><td> 전지 시스템 </td><td> 2B2-C75 </td><td></td></tr><tr><td> DMSO </td><td> 피셔 과학 </td><td> BP231-100 </td><td></td></tr><tr><td> DPBS </td><td> 시그마 알드리치 </td><td> D8537 </td><td></td></tr><tr><td> CaCl 2, MgCl 2 DPBS </td><td> 시그마ldrich </td><td> D8662 </td><td></td></tr><tr><td> Phorbol-myristate 아세테이트 (PMA) </td><td> 시그마 알드리치 </td><td> 79346-1MG </td><td></td></tr></tbody></table>

detection of nitric oxide and superoxide radical anion by electron paramagnetic resonance spectroscopy from cells using spin traps

낮은 농도에서 반응 질소 / 산소 종 (ROS / RNS)은 신호 전달, 세포 기능 조절에 중요한 역할을하고, 면역 반응하지만, 관계가없는 농도에서 세포 생존 능력 1, 2에 해로운입니다. 살아있는 시스템이 ROS 생성을 조절하는 내생과식이 항산화 방어 메커니즘으로 발전했지만, ROS는 산소의 정상적인 신진 대사의 부산물 자연으로 연속적으로 생산되며 단백질 기능의 손실, DNA의 절단, 또는 지방질의 결과 biomolecules의 산화 손상을 일으킬 수 peroxidation 3, 궁극적으로 세포 부상 또는 사망 4로 이어지는 산화 스트레스합니다. 초과 산화물 라디칼 음이온 (O 2 • -)는 같은 peroxynitrite와 급진 히드록실과 같은 생물 학적 시스템에 존재하는 것으로 알려진 가장 높은 산화 종의 주요 전구체이다. O 2의 생성 • - 그러므로 산화 버스트의 첫 징후 신호를하고, 전TS 감지 및 / 또는 생물 학적 시스템의 격리가 중요합니다. 이 예제에서는 O 2 •이 - polymorphonuclear neutrophils (PMNs)에서 생성되었다. phorbol-12-myristate-13-아세테이트 (PMA)와 chemotactic 자극을 통해, PMN는 O 2 •를 생성 - 니코틴 아데닌 dinucleotide 인산 (NADPH) 옥시 데이스 5 활성화를 통해. 세 isoforms으로 제공 질소 산화물 (NO) synthase는 inducible -,의 연결 및 내피-NOS, 또는 iNOS, nNOS 나 eNOS는 각각 어떠한 6 생산도하는 NADPH를 이용하여 L-시트룰린로 L-아르기닌의 전환 catalyzes로 . 여기서는 내피 세포에서 아니오를 생성 않습니다. 산화 스트레스 조건 하에서, 예를 들어 eNOS는 O 2 •로 NO 생산으로 전환할 수 - 헴 7 또는 공동 인자, tetrahydrobiopterin (BH 4) 8 산화로 인한 것으로 생각됩니다 uncoupling라는 프로세스에. 오직 몇 가지가있다하지만 생물 학적 시스템의 자유 래디 칼의 검출을위한 안정적인 방법은 특이성과 감도에 의해 제한됩니다. 스핀 트래핑은 일반적으로 자유 래디 칼의 식별에 사용되며 전자 상자 성체의 공명 (EPR) 분광학에 의해 감지 수있는 영구적인 스핀 adduct를 형성 스핀 트랩에 대한 급진의 추가 반응을 포함하고 있습니다. 다양한 급진 부가물가 생성되는 래디 칼을 식별하고 급진적인 생산 9 자연과 속도론에 관한 풍부한 정보를 제공할 수 사용될 수 특유의 스펙트럼을 나타냅니다. 주기적 nitrones, 5,5 - 디메틸-pyrroline-N-옥사이드, DMPO 10 phosphoryl - 치환 DEPMPO 11 및 에스테르 - 치환, EMPO 12 BMPO 13, 널리 스핀 트랩으로 고용되어 - 후자의 스핀 adduct - 함정는 O 2 • 위해 더 이상 반 목숨을 전시. 철 (II)-N-메틸-D-glucamine dithiocarbamate, 철 (MGD) 2 &lt;/ 서브&gt;는 일반적으로 adduct 형성과 스핀 adduct 14 높은 안정성의 높은 비율로 어떠한 인해를 잡을수 없다하는 데 사용됩니다.

Watch this Scientific Journal Video about Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps at JoVE.com

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

전자 상자 성체의 공명 (EPR) 분광학은 철 (II)-N-메틸-D-glucamine dithiocarbamate, 철 (MGD)를 사용하여 인간 neutrophils에서 소 대동맥 내피 세포와 초과 산화물 라디칼 음이온에서 질소 산화물을 감지하기 위해 고용되었다<sub&gt; 2</sub&gt;와 5,5 - 디메틸-1-pyroroline-N-산화물 각각 DMPO.

질소 산화물과 세포로부터 전자 상자 성체의 프로그램 공명 분광학에 의한 초과 산화물 음이온 급진적가 탐지 스핀 트랩을 사용하여

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

detection-nitric-oxide-superoxide-radical-anion-electron-paramagnetic

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Biology

18.8K 조회수.  The Ohio State University. 전자 상자 성체의 공명 (EPR) 분광학은 철 (II)-N-메틸-D-glucamine dithiocarbamate, 철 (MGD)를 사용하여 인간 neutrophils에서 소 대동맥 내피 세포와 초과 산화물 라디칼 음이온에서 질소 산화물을 감지하기 위해 고용되었다 2와 5,5 - 디메틸-1-pyroroline-N-산화물 각각 DMPO.

비디오: Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

전자 스핀 공명 산소 매핑에 대한 라이브 종의 마이크로 이미징

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

연구

생물학

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

C2C12 Myoblast 세포를 사용하여 Bioenergetic 프로필 실험

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each other at the biochemical level1. Nuclear magnetic resonance (NMR) spectroscopy is one of several technologies, including gas chromatography&#x2013;mass spectrometry (GC-MS), with considerable promise for such studies. Advantages of NMR are that it is suitable for untargeted analyses, provides structural information and spectra can be queried in quantitative and statistical manners against recently available databases of individual metabolite spectra2,3. In addition, NMR spectral data can be combined with data from other omics levels (e.g. transcriptomics, genomics) to provide a more comprehensive understanding of the physiological responses of taxa to each other and the environment4,5,6. However, NMR is less sensitive than other metabolomic techniques, making it difficult to apply to natural microbial systems where sample populations can be low-density and metabolite concentrations low compared to metabolites from well-defined and readily extractable sources such as whole tissues, biofluids or cell-cultures. Consequently, the few direct environmental metabolomic studies of microbes performed to date have been limited to culture-based or easily defined high-density ecosystems such as host-symbiont systems, constructed co-cultures or manipulations of the gut environment where stable isotope labeling can be additionally used to enhance NMR signals7,8,9,10,11,12. Methods that facilitate the concentration and collection of environmental metabolites at concentrations suitable for NMR are lacking. Since recent attention has been given to the environmental metabolomics of organisms within the aquatic environment, where much of the energy and material flow is mediated by the planktonic community13,14, we have developed a method for the concentration and extraction of whole-community metabolites from planktonic microbial systems by filtration. Commercially available hydrophilic poly-1,1-difluoroethene (PVDF) filters are specially treated to completely remove extractables, which can otherwise appear as contaminants in subsequent analyses. These treated filters are then used to filter environmental or experimental samples of interest. Filters containing the wet sample material are lyophilized and aqueous-soluble metabolites are extracted directly for conventional NMR spectroscopy using a standardized potassium phosphate extraction buffer2. Data derived from these methods can be analyzed statistically to identify meaningful patterns, or integrated with other omics levels for comprehensive understanding of community and ecosystem function.

A method for metabolite extraction from microbial planktonic communities is presented. Whole community sampling is achieved by filtration onto specially prepared filters. After lyophilization, aqueous-soluble metabolites are extracted. This approach allows for application of environmental metabolomics to trans-omics investigations of natural or experimental microbial communities.

핵 자기 공명 분광학을 사용하여 환경 Metabolomics를위한 낮은 밀도 Planktonic 커뮤니티에서 Metabolites의 농도

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

라이브의 검출<em&gt; 대장균</em&gt; O157 : PMA-qPCR에 의한 H7 셀

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

세포 내 칼슘의 두 광자 이미징<sup&gt; 2 +</sup&gt; 고립 된 쥐 대동맥의 취급 및 내피 세포에서 산화 질소 생산과 부드러운 근육 세포

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

본래 동맥의 내피 층에서 산화 질소와 활성 산소의 욕실 얼굴 인식

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

개발 및 특성화의<em&gt; 체외</em&gt; 미세 혈관 네트워크 및 내피의 정량적 측정 [칼슘<sup&gt; 2 +</sup&gt;]<sub&gt; 전</sub&gt; 및 질소 산화물 생산

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

민감한 면역를 사용하여 시토신의 수정 된 형태의 검출

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into nitrite and nitrate by interactions with various oxy-heme proteins and other still not well known pathways. The reverse process, reduction of nitrite and nitrate into NO had been discovered in mammals in the last decade and it is gaining attention as one of the possible pathways to either prevent or ease a whole range of cardiovascular, metabolic and muscular disorders that are thought to be associated with decreased levels of NO. It is therefore important to estimate the amount of NO and its metabolites in different body compartments &#8212; blood, body fluids and the various tissues. Blood, due to its easy accessibility, is the preferred compartment used for estimation of NO metabolites. Due to its short lifetime (few milliseconds) and low sub-nanomolar concentration, direct reliable measurements of blood NO in vivo present great technical difficulties. Thus NO availability is usually estimated based on the amount of its oxidation products, nitrite and nitrate. These two metabolites are always measured separately. There are several well established methods to determine their concentrations in biological fluids and tissues. Here we present a protocol for chemiluminescence method (CL), based on spectrophotometrical detection of NO after nitrite or nitrate reduction by tri-iodide or vanadium(III) chloride solutions, respectively. The sensitivity for nitrite and nitrate detection is in low nanomolar range, which sets CL as the most sensitive method currently available to determine changes in NO metabolic pathways. We explain in detail how to prepare samples from biological fluids and tissues in order to preserve original amounts of nitrite and nitrate present at the time of collection and how to determine their respective amounts in samples. Limitations of the CL technique are also explained.

Nitric oxide (NO) is an important signaling molecule in vascular homeostasis. NO production in vivo is too low for direct measurement. Chemiluminescence provides useful insight into NO cycle via measuring its precursors and oxidation products, nitrite and nitrate. Nitrite / nitrate determination in body tissues and fluids is explained.

화학 발광 법을 사용하여 생체 물질에서 산화 질소 통로에서 아질산염과 질산염, 대사 산물을 측정

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO&#8226; in vivo and in vitro, the real-time monitoring of NO&#8226; at the single-cell level is very challenging. The physiological or pathological effects of NO&#8226; are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO&#8226; are highly desirable. Recently, we expanded the pallet of NO&#8226; indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO&#8226;) probes (geNOps) that directly respond to cellular NO&#8226; fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO&#8226; signals in response to two different chemical NO&#8226;-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO&#8226; levels as compared with the inorganic NO&#8226; donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO&#8226; formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO&#8226; signals in diverse experimental setups.

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&amp;#8226;) Probes, the geNOps, for Real-time Imaging of NO&amp;#8226; Signals in Single Cells

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

유전자 인코딩 된 형광 산화 질소의 응용 프로그램 (• NO) 단일 세포에서 NO • 신호의 실시간 이미징을위한 프로브의 geNOps,

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...