이 작품은 건강의 국립 연구소 (CA142664)에 의해 지원되었다.

1. 카르복시 - H 2 DCFDA를 사용하여 ROS의 탐지 카르복시 - H 2 DCFDA이 아닌 형광하지만이 시약은 산화는 ROS의 면전에서, 그것은 녹색 형광됩니다. <ol><li> 사용하기 바로 전에, 살균 dimethylsulfoxide (DMSO) 또는 100 % 에탄올에 카르복시 - H 2 DCFDA의 신선한 재고 솔루션을 준비합니다. 당신의 염색 여러 해동 / 정지 사이클을 피하십시오. </li><li> HEPES로 세포를 씻어하면 소금 솔루션 (HBSS)을 버퍼 또는 식염수 (PBS)는 원래 매체의 흔적을 제거하는 인산 버퍼. </li><li> 염색과 세포 (및 제어 염료, CF 노트 섹션)로드합니다. 이 분석에서 우리는 Jurkat, 인간의 백혈병 세포 라인을 사용합니다. 감소 혈청 (2 %)와 정기적인 문화 매체에서 1μM의 최종 농도에서 카르복시 - H 2 DCFDA을 사용합니다. </li><li> 종래의 인큐베이터 (37 ° C 5 % CO2)에서, 어둠 속에서 30 분 동안 문화를 품어. 모든 않는 염료 솔루션을 폐기하십시오. </li><li> 카르복시를 제거- H 2 DCFDA는 매체가 포함된 HBSS 또는 PBS로 두 번 씻는다. 앞으로이 단계에서, 라이트로부터 세포를 보호합니다. </li><li> 카르복시 - H 2 DCFDA -로드 셀 선택의 약물을 포함하는 새로운 매체를 추가하고 원하는대로 알을 품다. 이 예를 들어 우리는 1 시간 H 2 O 2 (0.03 %)를 사용합니다. </li><li> 즉시 FL1 채널 (녹색 형광) 또는 형광 판 리더에 의해, 또는 형광 현미경을 사용하여 유동세포계측법하여 세포를 분석하여 ROS을 평가합니다. 형광은 녹색 형광에 적합한 여기 및 방출 파장을 사용하여 검색하실 수 있습니다. </li></ol> 참고 : 컨트롤이 카르복시 - H 2 DCFDA -로드 셀 및 치료 흠없는 치료 세포를 포함해야합니다. 카르복시 - H 2 DCFDA는 peroxides를 감지하는 것으로 알려져 있지만 다른 ROS에 의한 산화 수 있습니다. 이 시약은 또한 5, 기타 방법으로 산화 구별 제어 염료를 수정할 수 있습니다 - (및 6) - 카르복시 - 2 &#39;, 7&#39; - dichlorofluorescein의 디아 세 테이트 (카르복시 - DCFDA)은 따라서 시험에 포함되어야합니다. 2. 질산 산화물 (NO) 생산의 측정 당신은 Sulfanilamide 및 N - 1 - napthylethylenediamine의 dihydrochloride (NED) 솔루션, 아질산염 표준이 필요합니다. 이 분석은 Griess 분석이라고합니다. NED 솔루션 : 5 % 인산에 희석 sulfanilamide의 1 % 솔루션을 확인하십시오 : 무균 water.Sulfanilamide 솔루션 희석 N - 1 - napthylethylenediamine의 dihydrochloride의 0.1 % 솔루션을 확인하십시오. 아질산염 표준 : 멸균 매체 100μM로 0.1M 표준 재고 아질산 나트륨을 희석 같은 매체에서 시리얼 희석 해. 보관 조건 : 같은 상온에서 제조가 감독한 저장 화학 물질. 재구성하면, NED와 Sulfanilamide 솔루션은 어둠 속에서, 3 개월 최대 즉시 사용 4 ° C 이후에 저장됩니다. <ol><li> 96 잘 플레이트 문화 세포,사용은 각각의 조건에 대한 triplicates, 그리고 실험에 따라 적절한 컨트롤을 포함합니다. </li><li> 어떠한 생산을 유도하지 세포를 드셔보세요. 우리의 실험에서 우리는 우리의 세포를 치료 lipopolysaccharide (LPS) (100 NG / ML)와 재조합 IL - 4를 사용합니다. 이 프로토콜에서는, 우리는 RAW 264.7, 마우스 대식 세포의 세포 라인 (마우스 leukaemic 단핵구 대식 세포 세포 라인) 사용됩니다. </li><li> 분석의 날, 상온에 두 시약을 가지고. </li><li> 접시를 스핀, 휴대 supernatants를 수집하고 새로운 96 잘 판에 50μl를 전송할 수 있습니다. 표준 곡선을 만들기 위해 여러분의 표준 재고 솔루션의 희석 우물을 제한 준비합니다. </li><li> 각각의 샘플을 잘 제어하는​​ Sulfanilamide 솔루션의 50μl를 추가하고 잘 섞는다. </li><li> 어둠 속에서 10 분 동안 상온에서 부화. </li><li> 각각의 샘플을 잘 제어하는​​ N - 1 - napthylethylenediamine의 dihydrochloride 솔루션의 50μl를 추가하고 잘 섞는다. </li><li> 어둠 속에서 10 분 동안 상온에서 부화. </li><li> absorba를 측정즉시 520nm와 550nm 사이의 파장의 필터 플레이트 리더를 사용 제정신. </li></ol> 당신은 다른 접시 / 그릇 크기를 사용하는 경우, 각 솔루션과 샘플 표면에 뜨는 대한 1/1/1 볼륨을 사용합니다. 바이올렛 색상은 긍정적인 우물에 나타납니다. 표준 얻은 결과는 솔루션의 안정성을 확인 도움이 될 것입니다. 3. ROS 행동 탐지 : 산화 단백질 ROS의 생산을 식별하는 다른 방법은 단백질의 산화를 감지하여 최종 결과를 볼 수 있습니다. 사실, ROS 수정 글루타티온, 대부분의 세포에서 표현하고있는 항산화은 ROS에 대한 보호 역할을합니다. ROS는 이황화 다리를 통해 두 번째 글루타티온에 링크된되고 그 시스테인의 sulfhydryl (thiol) 그룹에서 감소 글루타티온 (GSH) 결과를 수정하여 산화를 따라. 이것은 (산화 단백질 GSSG) dimerized 단백질의 형성에 이르게. GSH는 효소의 환원 효소에 의해 글루타티온 GSSG의 수정을 통해 복원할 수 있습니다. GSSG / GSH 비율의 증가는 산화 스트레스를 반영합니다. 다음과 같은 분석은 이러한 산화 단백질의 탐지 및 부량을 기반으로합니다. 이 방법은 특정 ROS에 대한 선택은 아니지만 오히려 NO의 효과를 감지, H 2 O 2, O 2 -와 다른 ROS. 여기 발광 생물 신호를 사용하여 산화 (GSSG)와 감소 (GSH) 글루타티온의 총 양을 측정합니다. <ol><li> 평소처럼 96 잘 플레이트 (투명 / 화이트, 평평한 바닥)에 씨앗 당신의 세포를. 우리의 실험을 위해, 우리는 자기편 원시 264.7 및 A549 세포와 서스펜션 Jurkat 세포에 대해 잘마다 1x10 4 세포를 사용합니다. 당신의 세포의 크기에 따라 숫자가 조정 될 수 있습니다. 테스트 그들이 접시에 첨부할 수 있도록하기 전에 우리가 판 자기편 세포 하루 권장합니다. 충분히 웰스는 산화 및 감소 단백질 탐지뿐만 아니라 &quot;중간에만&quot;에 대한 준비하고 있어야컨트롤로 치료 세포. 사용은 모든 조건 triplicates. </li><li> 소요 시간에 대한 약물로 세포를 처리합니다. 여기 우리는 H 2 O 2 (5mM과 2.5mM 각각에서)와 Jurkat 세포와 1 시간을 위해 A549 세포를 치료. 원시 264.7 세포는 16 H.위한 LPS의 200ng/ml로 치료했다 이 배양 시간 동안 실온 (RT)에 모든 시약을 가지고. 더 이상 30 분 테스트를 수행하기 전에 모든 시약을 만듭니다. 아래의 표는 물론 당 시약의 볼륨을 보여줍니다. 가능한 경우, 그것은 검사하기 전에 줄이고 에이전트를 포함하고있는 미디어를 제거하는 것이 중요합니다. </li></ol><table border="1"><tbody><tr><td rowspan="2"></td><td colspan="2"> 자기편 세포 </td><td colspan="2"> 서스펜션 세포 </td></tr><tr><td> 총 GLU의 용해 </td><td> 산화 GLU의 용해 </td><td> 총 GLU의 용해 </td><td> 산화 GLU의 용해 </s&gt;를 tron​​g </td></tr><tr><td> NEM, 25mM </td><td> 없음 </td><td> 0.5μl </td><td> 없음 </td><td> 0.5μl </td></tr><tr><td> Luciferin - NT </td><td> 1 μl </td><td> 1 μl </td><td> 1 μl </td><td> 1 μl </td></tr><tr><td> 수동 용해 버퍼, 5 배 </td><td> 10μl </td><td> 10μl </td><td> 10μl </td><td> 10μl </td></tr><tr><td> 증류수 </td><td> 39.0μl </td><td> 38.5μl </td><td> 14μl </td><td> 13.5μl </td></tr><tr><td> 잘마다 최종 부피 </td><td> 50μl </td><td> 50μl </td><td> 25μl </td><td> 25μl </td></tr></tbody></table><ol start="3"><li> 자기편 세포와 우물에서 매체를 제거합니다. 서스펜션 세포와 우물에서 매체를 제거하지 마십시오. </li><li> 해당 우물로 축소 글루타티온 용해 시약이나 산화 글루타티온의 용해 시약을 추가하고 RT에서 5 분 흔들. </li><li> Luciferin 생성 시약 및 incubat 추가RT에서 30 분 E. </li><li> Luciferin 탐지 시약을 추가하고 RT에서 15 분 동안 품어. </li><li> 0.25-1 초당 잘 플레이트 리더 luminometer를 사용하여 통합시 발광 생물 신호를 읽어 보시기 바랍니다. </li></ol><table border="1"><tbody><tr><td></td><td> 자기편 세포 </td><td> 서스펜션 세포 </td></tr><tr><td> 물론 당 핸드폰 번호 </td><td> 1x10 4 </td><td> 1x10 4 </td></tr><tr><td> 물론 + 약물 당 세포 현탁액 </td><td> 3.4 전에 제거 100 μl, </td><td> 25μl, 삭제되지 않도록 </td></tr><tr><td> 감소 글루타티온의 용해 시약 </td><td> 50 μl </td><td> 25μl </td></tr><tr><td> 산화 글루타티온 용해 시약 </td><td> 50 μl </td><td> 25μl </td></tr><tr><td> Luciferin 생성 시약 </td><td> 50 μl </td><td> 50 μl </td></tr><tr><td> Luciferin 탐지 시약 </td><td> 100 μl </td><td> 100 μl </td></tr></tbody></table> 4. 대표 결과 : <img alt="그림 1" src="/files/ftp_upload/3357/3357fig1.jpg" /> 카르복시 - H2DCFDA 염료를 사용하여 ROS의 그림 1 탐지. H 2 O 2 처리된 Jurkat 세포는 (인간의 백혈병 세포 라인) 이외의 처리 세포에 비해했다. ROS는 유동세포계측법, O이 치료 세포가 컨트롤의 봉우리 (H 2 O 2 치료 세포를 산화 구별 염색 및 비 물들일에 비해 이동 H 2의 형광 피크 감지와 같은 녹색 fluoresces 카르복시 - H 2 DCFDA의 수정을 유도 카르복시 - H 2 DCFDA 물들일 - 대우 전지). 결과는 치료 세포에 ROS의 존재를 확인합니다. <img alt="그림 2" src="/files/ftp_upload/3357/3357fig2.jpg" /> 그림 2 탐지 OF Griess의 시약을 사용하여 어떠한이 없습니다. RAW 264.7 세포 (마우스 대식 세포)가 LPS와 IL - 4로 처리되었습니다. 중요한 NO 생산 증가는 치료 세포를 제어에 비해 처리 세포에서 발견되었다. <table border="1"><tbody><tr><td> 셀 라인 </td><td> 비 - 대우 </td><td> 치료 </td></tr><tr><td> 원시 264.7 </td><td> 13.0 </td><td> 8.3 </td></tr><tr><td> A549 </td><td> 21.6 </td><td> 10.5 </td></tr><tr><td> Jurkat </td><td> 5.2 </td><td> 2.8 </td></tr></tbody></table> ROS의 표 1 탐지 단백질의 산화를 중재. RAW 264.7 세포는 LPS, Jurkat 및 A549과 함께 치료를했다 (인간 폐암) 세포는 H 2 O 2 치료를했다. 비율이 감소 (GSH) / (GSSG) 글루타티온를 산화로 결과가 표현됩니다. 글루타티온 (GSH) / (GSSG)의 낮은 비율을 비교하여 처리 세포에서 발견되었습니다단백질이 더 많은 치료 세포에 산화다고 공개, 치료 세포를 제어합니다.

<ol>
	<li>Guzik, T. J., Korbut, R., Adamek-Guzik, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+and+superoxide+in+inflammation+and+immune+regulation.">Nitric oxide and superoxide in inflammation and immune regulation.</a> J Physiol Pharmacol. 54, 469-487 (2003).</li><li>Perl, A., Gergely, P., Banki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+T+cells+of+patients+with+systemic+lupus+erythematosus.">Mitochondrial dysfunction in T cells of patients with systemic lupus erythematosus.</a> Int Rev Immunol. 23, 293-313 (2004).</li><li>Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T., Mazur, M., Telser, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+and+antioxidants+in+normal+physiological+functions+and+human+disease.">Free radicals and antioxidants in normal physiological functions and human disease.</a> Int J Biochem Cell Biol. 39, 44-84 (2007).</li><li>Droge, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+in+the+physiological+control+of+cell+function.">Free radicals in the physiological control of cell function.</a> Physiol Rev. 82, 47-95 (2002).</li><li>Nakamura, K., Yube, K., Miyatake, A., Cambier, J. C., Hirashima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+CD4+D3-D4+membrane+proximal+extracellular+domain+for+the+inhibitory+effect+of+oxidative+stress+on+activation-induced+CD4+down-regulation+and+its+possible+role+for+T+cell+activation.">Involvement of CD4 D3-D4 membrane proximal extracellular domain for the inhibitory effect of oxidative stress on activation-induced CD4 down-regulation and its possible role for T cell activation.</a> Mol Immunol. 39, 909-921 (2003).</li><li>Los, M., Droge, W., Stricker, K., Baeuerle, P. A., Schulze-Osthoff, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+as+a+potent+activator+of+T+lymphocyte+functions.">Hydrogen peroxide as a potent activator of T lymphocyte functions.</a> Eur J Immunol. 25, 159-165 (1995).</li><li>Deem, T. L., Cook-Mills, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+cell+adhesion+molecule+1+(VCAM-1)+activation+of+endothelial+cell+matrix+metalloproteinases:+role+of+reactive+oxygen+species.">Vascular cell adhesion molecule 1 (VCAM-1) activation of endothelial cell matrix metalloproteinases: role of reactive oxygen species.</a> Blood. 104, 2385-2393 (2004).</li><li>Pacher, P., Beckman, J. S., Liaudet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+and+peroxynitrite+in+health+and+disease.">Nitric oxide and peroxynitrite in health and disease.</a> Physiol Rev. 87, 315-424 (2007).</li><li>Griendling, K. K., Sorescu, D., Lassegue, B., Ushio-Fukai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+protein+kinase+activity+and+gene+expression+by+reactive+oxygen+species+and+their+role+in+vascular+physiology+and+pathophysiology.">Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology.</a> Arterioscler Thromb Vasc Biol. 20, 2175-2183 (2000).</li><li>Loh, K., Deng, H., Fukushima, A., Cai, X., Boivin, B., Galic, S., Bruce, C., Shields, B. J., Skiba, B., Ooms, L. M., Stepto, N., Wu, B., Mitchell, C. A., Tonks, N. K., Watt, M. J., Febbraio, M. A., Crack, P. J., Andrikopoulos, S., Tiganis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+enhance+insulin+sensitivity.">Reactive oxygen species enhance insulin sensitivity.</a> Cell Metab. 10, 260-272 (2009).</li><li>Goldstein, B. J., Mahadev, K., Wu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+paradox:+insulin+action+is+facilitated+by+insulin-stimulated+reactive+oxygen+species+with+multiple+potential+signaling+targets.">Redox paradox: insulin action is facilitated by insulin-stimulated reactive oxygen species with multiple potential signaling targets.</a> Diabetes. 54, 311-321 (2005).</li></ol>

이 게시를 위해 Promega의 지원을 받았다.

같은 염증성 질환, 암, 국소 빈혈 / reperfusion하고, 또한 방사선 또는 화학 요법으로 치료 등 여러 병적인 상황 (즉, cisplatin) ROS의 생산 과잉을 유발. 따라서, 검출 및 ROS의 수준을 측정하는 여러 기본 사전 임상 및 임상 연구에 중요합니다. 그러나, ROS는 매우 짧은 절반 생활을하고 감지하는 복잡한 수 있습니다. 여기, 우리는 포유 동물 세포에 자유 라디칼 생산의 검출을 위해 정기적으로 사용하고 널...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> 5 - (그리고 - 6) - 카르복시 - 2 &#39;, 7&#39; - dichlorofluorescein의 디아 세 테이트 (카르복시 - DCFDA) </td><td> 분자 프로브 </td><td> C369 </td><td> 제어 </td></tr><tr><td> 카르복시 - H 2 DCFDA </td><td> 분자 프로브 </td><td> C400 </td><td></td></tr><tr><td> Sulfanilamide </td><td> 시그마 </td><td> S9251 - 100G </td><td></td></tr><tr><td> N - 1 - napthylethylenediamine의 dihydrochloride </td><td> 시그마 </td><td> N9125 - 10G </td><td></td></tr><tr><td> 아질산 기준 </td><td> 시그마 </td><td> 237213 - 100G </td><td></td></tr><tr><td> GSH / GSSG 저런 형광 분석 </td><td> Promega </td><td> V6612 </td><td> 계량, 산화 감소 또는 산화 / 글루타티온을 절감하기 위해 </td></tr></tbody>
</table>

production and detection of reactive oxygen species (ros) in cancers

반응 산소 종은 분자의 숫자를 포함 피해 DNA와 단백질 및 lipids (지질 peroxydation)을 RNA와 산화. 이러한 반응 분자가 산소를 포함하고 H 2 O 2 (과산화수소), NO (질산 산화물), O 2 포함 - (산화물 음이온), peroxynitrite (ONOO -), hydrochlorous 산성 (HOCl)과 히드록실를 라디칼 (OH -) . 산화 종뿐만 아니라 병적인 상황 (암 / 허혈성 reperfusion, neurologic 및 심혈관 pathologies, 전염성 질환, 염증성 질환 1, 면역 질환 2, 등 ...)에 따라뿐 아니라 세포의 대사 3으로 생리 (비 병적인) 상황 동안 생산됩니다 , 4. 사실, ROS는 (확산, 세포 활성화 5, 6, 7 마이 그 레이션 등.) 수많은 세포 신호 경로에서 중요한 역할을 재생합니다. ROS가 해로운 수 있습니다 (이것은 다음이라고합니다세포내 구획과 세포에 높은 금액에서 생산되는 &quot;산화와 nitrosative 스트레스&quot;) 일반적으로 초과 산화물의 dismutase (잔디)와 카탈 라 아제 (CAT), 글루타티온 퍼옥시데이즈 (GPX)와 보호 글루타티온 (GSH)와 같은 산화 억제제를 upregulating하여 ROS에 대응 무해한 분자 (예 : 물)에 위험한 자유 래디 칼을 변환하여. 비타민 C와 E는 ROS의 청소부 (산화 억제제)로 설명되었습니다. 자유 래디 칼은 낮은 금액 3 유용합니다. 대식 세포와 neutrophils - 중재 면역 반응은 바이러스, 병원균과 종양 증식 억제 8 NO의 생산 및 릴리스가 필요합니다. 어떠한 또한 다른 ROS와 반응하지 않으며 따라서, 또한 detoxifier (ROS의 청소)로 역할을하고 있습니다. 오랫동안 운동 9, 10 근육의 적응을위한 중요한 혈액 흐름을 조절하기 위해 선박에 마지막으로 어떠한 행위를하지 않습니다. 여러 출판물은 또한 ROS가 인슐린 SENS에 관련된 것을 증명itivity 11, 12. ROS 생산을 평가하는 많은 방법이 있습니다. 이 문서에서 우리는 몇 가지 간단하고 신속하고 저렴한 assays을 제안, 이러한 assays는 많은 출판물에 의해 검증되어 있고 정기적으로 포유 동물 세포에 ROS 또는 그 효과를 감지하는 데 사용됩니다. 이러한 assays 중 일부는 여러 ROS를 감지하는 동안, 다른 사람들은 단지 하나의 ROS를 감지합니다.

Watch this Scientific Journal Video about Production and Detection of Reactive Oxygen Species ROS in Cancers at JoVE.com

Production and Detection of Reactive Oxygen Species ROS in Cancers

여기 우리는 세포의 반응 산소 종의 존재를 테스트하고 평가하는 간단한 방법을 제안합니다.

암의 생산 및 반응 산소 종의 감지 (ROS)

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

production-and-detection-of-reactive-oxygen-species-ros-in-cancers

Research

JoVE Journal

Medicine

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

69.8K 조회수.  Baylor College of Medicine. 여기 우리는 세포의 반응 산소 종의 존재를 테스트하고 평가하는 간단한 방법을 제안합니다.

비디오: Production and Detection of Reactive Oxygen Species ROS in Cancers

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

급성 간염 모델 신청 : 시각화와 간장 Microcirculation의 혈류와 산소 소비 분석

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

연구

의학

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

암 치료 모니터링을위한 Cerenkov 발광 이미징 (CLI)

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

생물 검정으로 소 장간막 동맥과 정맥의 섹션을 분리하고 사용하면 소장에서 Vasoactivity을 테스트

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

와 전염성 해면상 뇌증 종 장벽 평가<em&gt; 체외</em&gt; 프리온 단백질 변환 분석

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T &#945;S and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of &#945;SD (disease-associated &#945;S) in these mice, an ELISA was designed to specifically quantify &#945;SD in sick mice. Analysis of the central nervous system in this mouse model showed the presence of &#945;SD mainly in the caudal brain regions and the spinal cord. There were no differences in &#945;SD distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of &#945;SD immunoreactivity using an antibody against Ser129 phosphorylated &#945;S by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of &#945;S. The propagation of &#945;SD, suggesting the involvement of a &#8220;prion-like&#8221; mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.

Detection of Disease-associated &amp;#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &amp;#945;-synuclein

An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated &#945;-synuclein (&#945;SD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated &#945;S form or the C-terminal part of the protein.

인간 A53T를 과발현 형질 전환 마우스의 뇌에 강화 된 ELISA에 의한 질병 관련 α-synuclein의 검출은 α-synuclein의 돌연변이

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) ...

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive astrogliosis. Reactive astrocytes exert both inflammatory and protective effects that inhibit and promote, respectively, neural repair. The mechanisms underlying the diverse functional properties of reactive astrogliosis are not well understood. Achieving a greater understanding of these mechanisms is critical to developing therapeutic strategies to treat the injured CNS. Here we demonstrate a method to trigger reactive astrogliosis in the adult mouse forebrain using a forebrain stab lesion. This lesion model is simple, reliable, and requires only a stereotaxic device and a scalpel blade to produce the injury. The use of stab lesions as an injury model in the forebrain is well established and amenable to studies addressing a broad range of neuropathological outcomes, such as neuronal degeneration, neuroinflammation, and disruptions in the blood brain barrier (BBB). Thus, the forebrain stab injury model serves as a powerful tool that can be applied for a broad range of studies on the CNS response to trauma.

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods.

반응성 신경교 증 트리거링<em&gt; 생체</em&gt; 전뇌 찔러 상해로

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

생체 외에서 그리고 Vivo에서 인간 세포, C. 선 충, 쥐에서 Mitophagy의 검출

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

VASP 인 산화를 측정 하 여 혈액에 산화 질소의 혈소판 기반 탐지

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

양성자 자기 공명 영상 및 조 영제로 산소를 사용 하 여 인간의 폐에 특정 통풍의 정량 매핑

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...

Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When deoxygenated, HbS can polymerize, forming rigid rods of hemoglobin, resulting in the sickling of red blood cells (RBCs). These sickled RBCs have significantly reduced deformability, causing vaso-occlusion, which leads to numerous SCD-related clinical complications, including pain, stroke, and organ damage. RBC deformability is also reduced by RBC dehydration, resulting in dense red blood cells that are more likely to sickle. To date, there is not a single widely available, rapid, and reproducible laboratory assay capable of predicting the disease severity or directly monitoring the treatment effects for novel, non-fetal hemoglobin inducing therapies. In this study, we describe a protocol to measure RBC deformability as a function of pO2 that allows for the quantitation of sickling behavior in SCD patients. Oxygen gradient ektacytometry measures RBC deformability, expressed as the elongation index (EI), as a function of pO2. RBCs are exposed to a fixed shear stress of 30 Pa during one round of deoxygenation and reoxygenation. Six readout parameters are produced. Of these, the point of sickling (PoS), defined as the pO2 at which maximum EI (EImax) shows a 5% decrease, and minimum EI during deoxygenation (EImin) are the most informative, reflecting an individual patient&#8217;s pO2 at which sickling starts and the minimal deformability of a patient&#8217;s red blood cells, respectively. PoS is associated with an individual patient&#8217;s hemoglobin affinity for oxygen, whereas EImin shows a strong correlation with fetal hemoglobin levels. We conclude that oxygen gradient ektacytometry is a promising technique to monitor the treatment of patients with SCD, as a biomarker for anti-sickling agents in clinical and preclinical trials, and an important tool to study sickling behavior of RBCs from individuals with SCD and sickle cell traits.

Here, we present oxygen gradient ektacytometry, a rapid and reproducible method to measure red blood cell deformability in samples from patients with sickle cell disease under controlled deoxygenation and reoxygenation. This technique provides a way to study red blood cell sickling and to monitor sickle cell disease treatment efficacy.

산소 그라데이션 Ektacytometry를 사용하여 제어 된 자동 탈산소 동안 병든의 특성

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When ...