이 작품은 PM 케인에 NIH R01 GM50322에 의해 지원되었다. 저자는 우리의 실험실에 대한 이러한 프로토콜을 작업에 박사 Rajini 라오, 효모 pHluorin 플라스미드를 제공하고 비례 pH 측정에 대한 조언 존스 홉킨스 대학 박사 글로리아 A. 마르티네즈 무 노즈 감사합니다.

1. BCECF-AM을 사용하여 생체 내에서 액포의 pH 측정 <ol><li> 밤새 원하는 매체에 측정 할 효모 균주의 50 ML 액체 문화를 성장. 목표는 (OD600 (600 nm에서 흡광도) 서스펜션 약 0.8 측정) 중반 로그 단계에서 세포를 가지고있다. </li><li> 원심 분리하여 펠렛 효모 세포. 성장 매체 이전에 무게 된 microcentrifuge 관에 전송 0.6 ㎖에 펠렛을 resuspend을. 60 초 동안 2,000 XG에서의 microcentrifuge에 다시 펠렛은 세포. 가능한 완벽하게 뜨는을 제거하고 세포 펠렛을 무게. 0.5 g / ㎖ (W / V)의 최종 농도에 펠렛을 resuspend을,이 볼륨의 문화는 약 200 밀리그램의 세포 펠렛 및 버퍼 200 μl를 제공합니다 400 μL의 최종 볼륨을 제공하기 위해 추가됩니다. </li><li> DMSO에 준비 12 밀리미터 주식에서 50 mm의 최종 농도에서 세포 현탁액에 BCECF-AM 추가 할 수 있습니다. 후 잘 입고 혼합30 분 동안 30 ° C에서 세포를 ubate. 흔들 플랫폼 또는 롤러 드럼. </li><li> 세포가 배양되는 동안, 보정 버퍼를 준비합니다. 50 MM의 HEPES (4 - (2 - 히드 록시 에틸)-1-piperazineethanesulfonic 산), 50 mM의 KCl을 50 mM의 NaCl을 0.2 M 암모늄 아세테이트, - 보정 버퍼는 50 mM의 MES를 ((N-모르) 에탄 산 2) 포함 10 MM 나트륨 아 지드, 10 MM 2-deoxyglucose 및 산도로 조절은 수산화 나트륨이나 염산의 보정 범위에 해당하는 값입니다. (주의 : 아 지드 화 나트륨은 매우 독성 및 취급에주의해야합니다.) 야생 형 세포에서의 pH 측정을 위해, 우리는 (6.5,하지만 더 많은 알칼리성 액포 산도가 예상 돌연변이에 대한 pH가 5.5에 여러 보정 혼합물을 준비 할 VMA 돌연변이), 우리는 추가, 높은 산도 버퍼를 준비합니다. </li><li> 나누어지는 2 15 ML 원뿔 튜브에 각각의 산도에 대한 보정 버퍼의 ML, 최종 110 μM의 모 넨신의 농도가 15 μM nigericin, R을 제공하는 모 넨신 (15 밀리미터 주)와 nigericin (2 MM의 주식을)를 추가espectively. 와류 믹서에 잘 섞는다. (주의 : 모 넨신 및 nigericin 모두가 독성 및주의하여 취급해야합니다.) </li><li> 의 microcentrifuge에서 2,000 XG에서 30 초 동안 원심 분리하여 BCECF-AM의 배양 시간이 완료되면 펠렛 세포 현탁액. 세포를 resuspend을 위와 포도당과 원심 분리기 부족한 성장 매체 1 ML입니다. 일단이 세척 단계를 반복 한 다음 포도당없이 성장 배지 200 μL에 펠렛을 resuspend을. 얼음에 위치. </li><li> 위의 준비 각 pH 보정 튜브에 세포 현탁액의 20 μl를 추가합니다. 30-60 분 동안 30 ° C에서 알을 품다. 롤러 드럼. </li><li> 교대 여기 파장 450 nm의 490 nm의 535 nm의에서 방출 파장에서 모두 측정 할 수 fluorimeter을 설정합니다. 30 ° C.에 샘플 챔버의 온도를 설정 큐벳의 혼합물의 연속 교반 모든 측정을 수행합니다. </li><li> 1 mM의 MES의 1.96 ML을 (세포의 성장 배지의 pH에​​ 따라 pH가 5 ~ 7로 조정 추가차 실험 설계). 큐벳에 세포 현탁액의 20 μl를 추가합니다. 형광 측정을 시작합니다. 우리는 지속적인 운동과 다른 실험 시간 포인트 데이터를 모두 수집합니다. 지속적인 운동 데이터의 경우, 우리는 5 분 동안 매 6 초 측정을 수행 한 후 50 mm의 최종 포도당 농도 포도당을 추가하고 5-10 분 동안 측정을 계속합니다. 단일 시간 지점 측정을 위해, 우리는 일반적으로 1에서 측정을 수행하고 5 분. 큐벳에 세포의 추가 후, 운동 측정에서와 같이 포도당을 추가하고 다른 측정 5 분 소요. 포도당 첨가 한 후. 이러한 추가는 변경 될 수 있으며, 추가 구성 요소 (억제제 등)도 추가 할 수 있습니다. </li><li> 실험 측정이 완료된 후, 30 ° C 배양에서 교정 튜브를 제거하고 fluorimeter 큐벳에 각각 전체 2 ㎖ 볼륨을 전송할 수 있습니다. 매 5 초마다 각 샘플에 대한 30 초 총에 (1.8 참조) 동일한 설정에서 형광을 측정합니다. </li><li>Microsoft Excel로 형광 데이터를 내보낼 수 있습니다. (우리 fluorimeter 들어,이 엑셀에 탭으로 구분 된 양식 가져 오기 텍스트와 같은 데이터의 수출이 필요합니다.) 각 교정 혼합물을 450 nm의 490 nm에서 형광의 비율을 계산하여 보정 곡선을 얻습니다. 형광 비율은 다음 보정 곡선을 얻을 수 산도 대 그려집니다. </li><li> 형광 비율을 실험 데이터를 변환하고 표준 곡선을 사용하여 pH를 계산합니다. 액포 pH는 다음 시간 (pH 변화의 역학)에 대한 플롯이나 다양한 조건 (그림 1)에서 또는 다른 돌연변이 균주에 비해 수 있습니다. </li></ol> 2. 효모 pHluorin을 사용하여 VIVO에서 세포질의 pH 측정 <ol><li> 표준 프로토콜에 의해 플라스미드 효모 pHluorin하여 원하는 효모 균주를 변환 및 보충 최소 배지 부족 우라실 (SC-우라실)에 형질 전환을위한 선택합니다. </li><li> 중반 로그 단계 (OD에 SC-유라의 변형 세포의 50 ML 액체 문화를 성장 <suB&gt; 600 = 0.8 이하). </li><li> 액포 산도하지만, 세포질 pH 측정, 일반적으로 pH를 6-8 적합한 pH 범위에 버퍼에 설명 된대로 교정 표준을 준비합니다. 보정 버퍼 나누어지는 2 ㎖는 몇 15 ML 원뿔 튜브에 원하는 산도를 조정했습니다. (1.4) 위에서 설명한대로 모 넨신 및 nigericin를 추가하고 잘 섞는다. </li><li> 위에서 설명한대로 원심 분리하여 세포를 수확하고, 성장 배지 600 μL에 펠렛을 resuspend을. 30 초 동안 5,000 rpm으로 원심 분리하여 무게 microcentrifuge 관 및 펠렛 세포에 전송합니다. 다시 포도당 (SC-우라실, 포도당), 펠렛 세포없이 성장 매체 1 ML에서 펠렛을 resuspend을 반복하십시오. 최종 원심 분리 한 후, 같은 철저 가능한 상층 액을 제거하고 세포 펠렛을 무게. 아니 포도당과 SC-우라실 0.5 g / ML의 최종 농도로 세포를 resuspend을. </li><li> 보정 버퍼의 각 튜브에 세포 현탁액의 20 μl를 추가하고 와류 믹서에 잘 섞는다. 3 품다0 ° ~ 60 분 동안 회전하는 드럼 로터 C. </li><li> 파장 405 및 485 nm의 여기 및 508 ㎚의 발광 파장에 대한 fluorimeter을 설정합니다. BCECF 측정을 위해 위에서 설명한대로 하나의 시점이나 운동 측정 및 혈당의 추가로 진행합니다. </li><li> 보정 샘플의 형광을 측정하고 BCECF (1.10-1.11 참조)로 보정 곡선을 생성합니다. 형광 비율 대 산도의 줄거리는 대부분의 세포질 pH 측정 (pH가 6.0-8.0) 9의 범위에서 선형이기 때문에, 실험 형광 비율 측정은 쉽게 산도로 변환됩니다. </li></ol>

<ol>
	<li>Casey, J. R., Grinstein, S., Orlowski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensors+and+regulators+of+intracellular+pH.">Sensors and regulators of intracellular pH.</a> Nat. Rev. Mol. Cell Biol. 11, 50-61 (2010).</li><li>Orij, R., Brul, S., Smits, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+pH+is+a+tightly+controlled+signal+in+yeast.">Intracellular pH is a tightly controlled signal in yeast.</a> Biochim. Biophys. Acta. 1810, 933-944 (2011).</li><li>Diakov, T. T., Kane, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+vacuolar+proton-translocating+ATPase+activity+and+assembly+by+extracellular+pH.">Regulation of vacuolar proton-translocating ATPase activity and assembly by extracellular pH.</a> J. Biol. Chem. 285, 23771-23778 (2010).</li><li>Martinez-Munoz, G. A., Kane, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vacuolar+and+plasma+membrane+proton+pumps+collaborate+to+achieve+cytosolic+pH+homeostasis+in+yeast.">Vacuolar and plasma membrane proton pumps collaborate to achieve cytosolic pH homeostasis in yeast.</a> J. Biol. Chem. 283, 20309-20319 (2008).</li><li>Tarsio, M., Zheng, H., Smardon, A. M., Martinez-Munoz, G. A., Kane, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+loss+of+Vph1+protein-containing+vacuolar+ATPases+(V-ATPases)+for+overall+cellular+pH+homeostasis.">Consequences of loss of Vph1 protein-containing vacuolar ATPases (V-ATPases) for overall cellular pH homeostasis.</a> J. Biol. Chem. 286, 28089-28096 (2011).</li><li>Plant, P. J., Manolson, M. F., Grinstein, S., Demaurex, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+mechanisms+of+vacuolar+acidification+in+H(+)-ATPase-deficient+yeast.">Alternative mechanisms of vacuolar acidification in H(+)-ATPase-deficient yeast.</a> J. Biol. Chem. 274, 37270-37279 (1999).</li><li>Ali, R., Brett, C. L., Mukherjee, S., Rao, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+sodium/proton+exchange+by+a+Rab-GTPase-activating+protein+regulates+endosomal+traffic+in+yeast.">Inhibition of sodium/proton exchange by a Rab-GTPase-activating protein regulates endosomal traffic in yeast.</a> J. Biol. Chem. 279, 4498-4506 (2004).</li><li>Miesenbock, G., De Angelis, D. A., Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+secretion+and+synaptic+transmission+with+pH-sensitive+green+fluorescent+proteins.">Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.</a> Nature. 394, 192-195 (1998).</li><li>Brett, C. L., Tukaye, D. N., Mukherjee, S., Rao, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yeast+endosomal+Na+K+/H++exchanger+Nhx1+regulates+cellular+pH+to+control+vesicle+trafficking.">The yeast endosomal Na+K+/H+ exchanger Nhx1 regulates cellular pH to control vesicle trafficking.</a> Mol. Biol. Cell. 16, 1396-1405 (2005).</li><li>Owen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+spectrum-shifting+intracellular+pH+probes+5'(and+6')-carboxy-10-dimethylamino-3-hydroxyspiro[7H-benzo[c]xanthene-7,+1'(3'H)-isobenzofuran]-3'-one+and+2',7'-biscarboxyethyl-5(and+6)-carboxyfluorescein.">Comparison of spectrum-shifting intracellular pH probes 5'(and 6')-carboxy-10-dimethylamino-3-hydroxyspiro[7H-benzo[c]xanthene-7, 1'(3'H)-isobenzofuran]-3'-one and 2',7'-biscarboxyethyl-5(and 6)-carboxyfluorescein.</a> Anal. Biochem. 204, 65-71 (1992).</li><li>Dechant, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytosolic+pH+is+a+second+messenger+for+glucose+and+regulates+the+PKA+pathway+through+V-ATPase.">Cytosolic pH is a second messenger for glucose and regulates the PKA pathway through V-ATPase.</a> Embo J. 29, 2515-2526 (2010).</li><li>Gustavsson, M., Barmark, G., Larsson, J., Muren, E., Ronne, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomics+of+monensin+sensitivity+in+yeast:+implications+for+post-Golgi+traffic+and+vacuolar+H+-ATPase+function.">Functional genomics of monensin sensitivity in yeast: implications for post-Golgi traffic and vacuolar H+-ATPase function.</a> Mol. Genet. Genomics. 280, 233-248 (2008).</li><li>Kovac, L., Bohmerova, E., Butko, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionophores+and+intact+cells.+I.+Valinomycin+and+nigericin+act+preferentially+on+mitochondria+and+not+on+the+plasma+membrane+of+Saccharomyces+cerevisiae.">Ionophores and intact cells. I. Valinomycin and nigericin act preferentially on mitochondria and not on the plasma membrane of Saccharomyces cerevisiae.</a> Biochim. Biophys. Acta. 721, 341-348 (1982).</li><li>Braun, N. A., Morgan, B., Dick, T. P., Schwappach, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yeast+CLC+protein+counteracts+vesicular+acidification+during+iron+starvation.">The yeast CLC protein counteracts vesicular acidification during iron starvation.</a> J. Cell Sci. 123, 2342-2350 (2010).</li><li>Orij, R., Postmus, J., Beek, T. e. r., Brul, A., S, G. J., Smits, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+cytosolic+and+mitochondrial+pH+using+a+pH-sensitive+GFP+derivative+in+Saccharomyces+cerevisiae+reveals+a+relation+between+intracellular+pH+and+growth.">In vivo measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in Saccharomyces cerevisiae reveals a relation between intracellular pH and growth.</a> Microbiology. 155, 268-278 (2009).</li><li>Zhang, Y. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+ergosterol+in+V-ATPase+function+underlies+antifungal+activity+of+azole+drugs.">Requirement for ergosterol in V-ATPase function underlies antifungal activity of azole drugs.</a> PLoS Pathog. 6, e1000939 (2010).</li><li>Brett, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+reveals+the+vacuolar+pH-stat+of+Saccharomyces+cerevisiae.">Genome-wide analysis reveals the vacuolar pH-stat of Saccharomyces cerevisiae.</a> PLoS One. 6, e17619 (2011).</li><li>Roberts, C. J., Raymond, C. K., Yamashiro, C. T., Stevens, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+studying+the+yeast+vacuole.">Methods for studying the yeast vacuole.</a> Methods Enzymol. 194, 644-661 (1991).</li><li>Chan, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+V-ATPase+proton+transport+reveal+uncoupling+functions+of+tether+linking+cytosolic+and+membrane+domains+of+V0+subunit+a+(Vph1p).">Inhibitors of V-ATPase proton transport reveal uncoupling functions of tether linking cytosolic and membrane domains of V0 subunit a (Vph1p).</a> J. Biol. Chem. 287, 10236-10250 (2012).</li><li>Johnson, R. M., et al. <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+inhibitors+of+vacuolar+proton-translocating+ATPase+pumps+in+yeast+by+high-throughput+screening+flow+cytometry.">Identification of inhibitors of vacuolar proton-translocating ATPase pumps in yeast by high-throughput screening flow cytometry.</a> Anal. Biochem. 398, 203-211 (2010).</li></ol>

저자는 공개 할 관심 없음 충돌이 없습니다.

우리의 pH 항상성 측면의 숫자를 해결하기 위해이 프로토콜을 이용했다. 예를 들어, 우리는 4,5 야생 형과 V-ATPase의 결핍 돌연변이 세포의 세포질과 산도 반응을 비교했다. 우리는 또한 포도당 3 액포의 pH 응답에 변경된 성장 조건, 특히 세포 산도의 효과를 조사했다. 중요한 것은, 우리가 관찰 응답은 양적 pH 측정의 다른 방법과와 양성자 펌프의 변경된 활동을 설명하는 생화 학적 ?...

의 pH 항상성은 모든 생물 1,2 역동적이고 고도로 통제 프로세스입니다. 생화학 적 과정은 밀접하게 산도에 의해 규제되고, 세포 환경은 주민 효소의 최적 활동을 할 수 있도록 좁은 산도 범위로 조정됩니다. 그러나 세포의 pH 항상성은 환경 산도, 대사 변화, 특정 신호 전달 경로의 급격한 변화에 도전 할 수 있습니다. 또한, 세포 내 pH는 중요한 신호로 자신을 제공 할 수 있습니다. 마지막으로, 많은 세포 소기관이 세포 소기관 특정 기능에 대한 주변의 세포질에서 독특하고 필수적인 lumenal의 pH 값을 유지합니다. 높은 진핵 생물 2의 pH 항상성 메커니즘의 효모 인 Saccharomyces cerevisiae의 주가는 숫자입니다. 리소좀 / endocytic 통로의 산성 세포 소기관으로, pH는 주로 많은 exchan와 협력하여 행동, 고도 보존 액포 양성​​자 translocating ATPase의 (V-ATPase의)에 의해 제어됩니다산도 기울기에 따라 제르. 모든 진핵 세포는 양성자 수출 메커니즘이 있습니다. 에 곰팡이와 식물, 플라즈마 막에서 두 번째, 독특한 프로톤 펌프, Pma1, 수출 신진 대사 양성자와는 세포질 pH와 세포막 전위의 주요 결정 요인이 될 것으로 생각됩니다. S.의 유전 적 유연성 cerevisiae의 및 상업적 중요성은, 그것의 pH 항상성이 공부를위한 매우 중요하고 흥미로운 모델을 만들었습니다. 세포 기관 산성화의 주요 드라이버 일뿐만 아니라, V-ATPases 매우 효소를 통제하고 우리의 실험실은 V-ATPase의 규제 메커니즘을 이해에 관심이 있습니다. 이 목표를 향해, 우리는 액포와 세포질의 pH 생체 pH 측정에 사용하고있다 : 1) 돌연변이의 효과 타협 V-ATPase의 활성을 조사하기 위해 포도당 박탈 및 readdition 2로 변경 세포 조건)에 대한 응답을 모니터링하는 데, 3) COOR를 탐험세포 소기관 및 플라즈마 막 양성자 펌프 3-5 조율 활동. 이러한 실험은 효모 세포에서 사용할 의무가 강력한 비례 산도 지표의 개발을 통해 가능하게되었다. . 포유 동물 세포의 세포질 산도를 측정하기 위해 널리 사용되었습니다, 효모 액포에 축적 - 플랜트 등 첫번째 BCECF ((6) carboxyfluorescein 2&#39;7&#39; - 비스 - (2 - 카복시 에틸) -5) 것으로 나타났다 대신 세포질 6. BCECF 현지화의 차이는 BCECF-AM (BCECF의 아세 톡시 메틸 에스테르) 및 액포 유지 6에서 아세 톡시 메틸 에스테르의 분열에 대한 가능성이 책임 액포에있는 많은 가수 분해 효소에 기인하고있다. 알리 등 7. 더 BCECF를 사용하여 액포의 pH 측정을 개발하고 형광 플레이트 리더 형식으로 이러한 측정을 적응. 브렛 등. 플라스미드 매개 R을 표현하여 효모의 세포질 산도를 측정하는 수단으로 효모 pHluorin 소개효모 특이 적 프로모터 9 통제 atiometric 산도에 민감한 GFP 8. BCECF 효모 pHluorin 모두의 여기 스펙트럼은 산도에 민감, 그래서 그들은 하나의 발광 파장에서 측정 된 두 여기 파장,에서 형광의 비율은 pH가 8,10의 측정을 제공하는 비례의 pH 지표로 사용됩니다. 이러한 효모 액포와 세포질 pH 센서는 모두 단일 셀 및 인구 기반 측정에 사용되었습니다. 단일 세포 측정은 6,11 형광 현미경 및 이미지 분석에 의해 수행됩니다. 두 파장에서의 액포 또는 세포질 형광 각 셀에 대해 측정됩니다. 인구 기반의 측정은 적절한 형광 기능을 갖춘 마이크로 플레이트 리더 중 하나 또는 fluorimeter에서 수행됩니다. 그것은 죄수 중에 이러한 포도당 같은 구성 요소의 추가에 대한 쉬운 접근을 제공하기 때문에 우리는 일반적으로 fluorimeter 우리의 측정을 수행 한tinuous 운동 측정. 액포와 세포질의 pH 측정을위한 우리의 현재 실험실 프로토콜은 아래에 나열되어 있습니다; 둘은 쉽게 마이크로의 분석에 적용됩니다.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Spectrofluorometer</td>
 <td>Horiba Jobin Yvon</td>
 <td>Model Fluoromax-4</td>
 <td>Temperature control and stirring capability are desirable.</td>
 </tr>
 <tr>
 <td>BCECF-AM</td>
 <td>Invitrogen/Molecular Probes</td>
 <td>B1150</td>
 <td>Prepare a 12 mM stock in dry DMSO, store as aliquots at -20 &deg;C </td>
 </tr>
 <tr>
 <td>monensin</td>
 <td>Sigma</td>
 <td>M5273</td>
 <td>Toxic. </td>
 </tr>
 <tr>
 <td>nigericin</td>
 <td>Sigma</td>
 <td>N7143</td>
 <td>Toxic. </td>
 </tr>
 <tr>
 <td>MES</td>
 <td>Sigma</td>
 <td>M8250</td>
 <td></td>
 </tr>
 </tbody>
</table>

measurement of vacuolar and cytosolic ph in vivo in yeast cell suspensions

액포와 산도 세포질이 높은 효모 세포의 조절과 전체의 pH 항상성에 중요한 역할을 차지한다. 우리는 공포 또는 세포질로 지역화 산도에 민감한 형광 물질을 사용하여 생체 내에서의 pH 비례 측정을위한 프로토콜을 설명합니다. 액포 pH는 그 아세 톡시 메틸 에스테르 형태로 세포에 도입하면 효모 공포에 지역화 BCECF를 사용하여 측정됩니다. 세포질 pH는 효모 발기인, 효모 pHluorin의 통제하에 표현 산도에 민감한 GFP로 측정됩니다. fluorimeter에서 효모 세포 현탁액의 형광 비율을 측정하는 방법이 설명되어 있습니다. 이러한 프로토콜을 통해 다른 조건 하에서, 또는 다른 효모 돌연변이의 pH 단일 시점의 측정이 비교되었고 시간이 지남에 따라 산도의 변화를 모니터링하고 있습니다. 이 방법은 높은 처리량 실험 형광 플레이트 리더 형식에 맞게 조정되었습니다. 다른 AP에 비례 pH 측정의 장점현재 사용 가능한 실험 문제 및 해결하고, 이러한 기술의 향후 사용에 대한 전망의 접근 방법도 설명합니다.

50 밀리미터 MES pH를 5로 버퍼링 된, 그림 1은 다양한 매체에서 자란 야생 형 효모 세포 (YEPD 효모 추출물 - 펩톤 - 덱스 트란)에서 얻은 액포의 pH 데이터를 제공합니다. 배지의 pH는 특히 최소 배지 하룻밤 성장하는 동안 매우 극적으로 변화 할 수 있기 때문에 우리는 종종 버퍼 매체에 세포를 성장하고, 우리는 성장 배지의 pH가 액포의 pH 응답하기 3에 영향을 미칠 수있는 것으로 ?...

Watch this Scientific Journal Video about Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions at JoVE.com

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

액포와 세포질 pH는 라이브 효모 측정 할 수있다 (<em&gt; S. cerevisiae의</em&gt;) 특정 세포 구획에 국한 비례 형광 염료를 사용하여 세포. 우리는 효모의 공포에 지역화 BCECF-AM, 그리고 세포질 비례 산도에 민감한 GFP (효모 pHluorin)와 세포질 산도와 액포 산도를 측정하기위한 절차를 설명합니다.

액포와 세포질의 pH 측정<em&gt; 생체 내</em&gt; 효모 세포 현탁액의

Vacuolar and  cytosolic pH are highly regulated in yeast cells and occupy a central role in  overall pH homeostasis. We describe  protocols for ...

measurement-vacuolar-cytosolic-ph-vivo-yeast-cell

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JoVE Journal

Biology

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

19.8K 조회수.  SUNY Upstate Medical University. 액포와 세포질 pH는 라이브 효모 측정 할 수있다 ( S. cerevisiae의) 특정 세포 구획에 국한 비례 형광 염료를 사용하여 세포. 우리는 효모의 공포에 지역화 BCECF-AM, 그리고 세포질 비례 산도에 민감한 GFP (효모 pHluorin)와 세포질 산도와 액포 산도를 측정하기위한 절차를 설명합니다.

비디오: Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

내부 핵심 현미경에 의해 골격근의 생체내 미세 순환 측정

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

연구

생물학

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

단일 및 다세포 상황에서 Invadopodia로 인한 세포 외 기질 Proteolysis의 양적 측정

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.

Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.

공 초점 현미경을 사용하여 살아있는 세포에서 세포 내 이입과 세포질 구획 동시 pH 측정

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

포유 동물 세포에서 헴 합성 레벨 측정

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

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

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

유동 세포 계측법에 의한 신장 상피 세포에서 세포 내 칼슘 측정

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

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

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

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

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

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

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

고해상도 이미징 및 개인 별이 미세 소관 역학의 분석 신진 효모에서

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

효모의 4D 현미경 검사법

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

효 모에 Polyglutamine 독성 분석 실험을 사용 하 여 퓨전 파트너에 형광 단백질의 영향

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

그대로 꼬마 선 충 에 산소 소비 속도의 측정

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...