We thank Dr. Gilles Dupuis for critical reading of the manuscript. 
Funding
This work was supported by the Canadian Institutes for Health Research, grant # MOP-126173. C.M.D is a member of the Fonds de la Recherche en Sant&#233; (FRSQ)-funded Centre de Recherche Clinique &#201;tienne LeBel.

셀룰러 산도 교정을위한 해결책 1. 준비 <ol><li> 교정에 사용되는 5 용액을 제조 5 개의 분리 50 ㎖ 원추형 튜브에 이하의 화합물을 추가 <ul><li> 염화나트륨 (1 M NaCl을 1 ㎖) (1 M NaCl을 10 ㎖의 H 2 0 = 0.58 g) </li><li> 의 KCl은 (7,425 1 M의 KCl ㎖) (1 M의 KCl은 10 ㎖의 H 2 0에서 825 g을 =) </li><li> 포도당 (1 M D-글루코스의 1 ㎖) (1M D-글루코스 = 10 밀리리터 H 1.80 g이 0) </li><li> MgS0 4 (0.05 1 M 망초의 ML) (1 M 망초 10 ㎖의 H 2 0 4 = 1.20 g) </li><li> 20 mM의 HEPES (1M HEPES 1 ㎖) (10 ㎖의 H 2 0 1 M HEPES는 = 2.38 g) </li><li> 염화칼슘 (1M 염화칼슘 0.05 ㎖) (10 ㎖의 H 1 M 염화칼슘 = 1.47 G 2 0) 두 번 증류 된 H 2 O (DDH 2 O)의 모든 솔루션을 준비합니다. </li></ul></li><li> DDH 2 O 35 ㎖로 각각의 솔루션을 완료하고 그렇게 할 때까지 자석 교반기로 혼합lution가 달성된다. </li><li> 5.5, 6.0, 6.5, 7.0 및 7.5의 pH 값을 얻기 위해 1 N KOH 또는 1 N HCl로 각 용액의 pH를 조정합니다. 이어서, 50 ㎖의 최종 부피의 탈 이온수를 사용하여 볼륨을 조절한다. </li><li> 측정 5 nigericin의 MG 및 원액 nigericin 10 밀리미터를 준비하는 무수 EtOH 670 μL에 추가합니다. 주의 : nigericin이 높은 독성과 큰 관심 (장갑, 보호 안경 및 마스크)로 처리해야합니다. nigericin가 완전히 용해 될 때까지 반전하여 솔루션을 섞는다. </li><li> 각각의 보정 용액에 10 mM의 nigericin (최종 농도 10 μM)의 0.05 ML을 추가합니다. Nigericin은 세포막 투과성을 증가시키고, 세포의 K + 탈분극 농도의 존재 하에서 세포 내 및 세포 외 pH를 평형화 원인 이온 운반체이다. </li><li> 교정 솔루션은 4 ° C에서 1개월까지 저장할 수 있습니다 </li></ol> 2. 휴대 준비 <ol><li> 생물학적 보안 시스템에서35mm의 Y 후드, 씨 세포 X 10mm 배양 접시 10 % 소 태아 혈청과 항생제가 보충 된 성장 배지 2 ㎖에. 24 시간 후에 약 50 % 합류점을 보장하기 위해 필요한 셀의 양을 사용한다. 주 : 우리의 실험에서, 우리는 HT-1080 세포주 및 플레이트 배양 접시 당 1 × 10 5 세포를 사용합니다. </li><li> 교정에 사용되는 실험 및 플레이트 (5 건) 문화 요리에 필요한 배양 접시의 수를 결정합니다. </li><li> 최소 24 시간 동안 37 ° C / 5 % CO 2 배양기에 넣고 배양 접시. </li></ol> pH를 감지 프로브 3. 준비 <ol><li> DDH 2 0에서 형광의 pH 프로브 8-hydroxypyrene-1, 3,6 trisulfonic 산, 삼 나트륨 염 (HPTS / pyranine)의 1M 주식 솔루션을 준비합니다. 이 솔루션은 실온에서 저장 빛으로부터 보호되어야한다. </li><li> 는 thereâ라고, (및-6) - 카르 복실 seminaphthorhodafluor 아세 톡시 메틸 에스터 아세테이트 (C-SNARF-1 AM - 1 ~ 5 ㎜의 재고 솔루션을 준비합니다따고 SNARF-1) DMSO 1.76 ㎖에 SNARF-1의 1 MG를 용해하여. 철저 반복 피펫으로 혼합. SNARF-1 솔루션은 -20 ° C에서 보관하고 빛으로부터 보호되어야한다. </li></ol> 4. 셀 라벨 <ol><li> 열 여섯 시간 라이브 세포 이미징 전에 완전 배지 2 ㎖를 포함하는 각 배양 접시에 1M HPTS 원액의 2 μL (최종 농도 1 ㎜)를 추가합니다. </li><li> 배양기에 배양 접시를 돌려줍니다. HPTS 프로브 따라서 라벨에 프로브는 세포 작용에 의해 흡수되어야한다 소기관, 세포 impermeant입니다. 이 조건은 HPTS와 세포 배양 혈청이 첨가 된 배지에서 수행해야합니다. </li><li> HPTS와 배양 후, 멸균 PBS로 두 번 세포를 세척하고 적어도 2 시간 동안 무 혈청 배지 2 ㎖를 추가합니다. SNARF -1 인큐베이션 혈청은 프로브의 흡수를 방지 할 수 있으므로 무 혈청 배지에서 수행 될 필요가있다. </li><li> AF를 얻기 위해 1 ㎜ SNARF-1 원액의 10 μl를 추가합니다원고 판 5 μM의 농도와 세포가 희미한 빛 아래에서, 37 ° C에서 20 분 동안 배양 할 수 있습니다. C-SNARF-1 농도와 배양 시간은 세포의 종류에 따라 다를 수 있습니다. </li><li> 부화의 끝에서, 멸균 PBS에 두 번 세포를 세척하고 무 혈청 배지 2 ㎖를 추가합니다. </li></ol> 산도 변조기 5. 추가 <ol><li> 이 방법은 세포에 pH 변화를 감지 할 수 있는지 여부를 평가하기 위해, 세포 내 산도를 조절하는 다른 시약을 사용할 수 있습니다 : <ul><li> Bafilomycin A1, V-ATPase의 5 특정 억제제 </li><li> NA + / H의 + 교환기 이소 형 1 (NHE-1) ~ (6) </li><li> NH 4 망할 CIA, 세포 내 pH가 7 증가 약한베이스 </li></ul><ol><li> 위에서 언급 한 시약의 주식 솔루션을 준비 : <ul><li> Bafilomycin A1 : 100 μM의 원액을 얻기 위해 DMSO 160 μL에 bafilomycin A1의 10 μg을 추가 </li><li> EIPA :는 obtai 메탄올 1.67 ml로 EIPA의 25 밀리그램을 추가50 mm의 최종 농도 NA </li><li> NH4Cl 등 : 1 mM의 최종 농도를 구하는 DDH 2 O 10ml에 염화 암모늄 0.53 g을 용해. </li></ul> 참고 : 모든 주식 솔루션은 -20 ° C에서 보관하고 빛으로부터 보호되어야한다 bafilomycin의 A1을 제외하고 4 ° C에 보관해야합니다. </li><li> 별도의 배양 접시에서, 아래에 설명 된대로 시약 중 하나를 추가합니다 : <ul><li> 100 μM의 bafilomycin A1의 2 μL (최종 농도 100 NM) </li><li> 50 mM의 EIPA 1 μL (최종 농도 25 μM) </li><li> 1M NH4Cl 등 (최종 농도 20 ㎜)의 40 μL </li></ul></li><li> 37 ℃에서 30 분 동안 세포를 품어 </li></ol></li><li> 이미지 수집을 진행합니다. </li></ol> 6. 라이브 세포 이미징 현미경의 사양 및 취득 설정 <ol><li> 사용하여 라이브 세포 이미징을 수행 40X 목적은 스펙트럼 반전 스캐닝 공 촛점 microsco에 장착퍼가기 전동 XY 스테이지, 환경 제어 (온도, 습도, CO 2) 단계 인큐베이터에서 유리 바닥 문화 요리를 수용하는 챔버를 탑재. </li><li> 여기에 사용 된 공 초점 악기 (올림푸스 FV1000)가 장착되어 있습니다 : <ul><li> 다이오드 레이저 405 nm의 </li><li> 40 mW의 아르곤 레이저 (458 nm의, 488 nm의, 515 nm의) </li><li> 헬륨 네온 레이저 (543 nm의) </li><li> 형석 10X 목적, NA0.4 </li><li> LUCPLFLN 40X 목적, NA0.60 </li><li> PLAPONSC PLAN APO 60X 오일 목적, NA1.4 </li><li> 다이크로 익 미러 DM 405/458/488/543 </li><li> SDM (560) 및 SDM (640), 다이크로 익 필터 </li><li> BA655-755 차단 필터 </li><li> 빔 스플리터 BS20/80 </li><li> 이미지 수집 및 분석을위한 데이터 분석 소프트웨어, </li></ul></li><li> 무대 인큐베이터가 37 ° C로 예열하고 현미경이 설정되면, 이전에 촛점 무대 영상 실에 HPTS와 SNARF-1 배양 배양 접시를 놓습니다. </li><li>관심 영역을 위치 따고, 소프트웨어 취득 설정도 1을 조정한다. <ul><li> 두 개의 가상 채널 세트를 설정합니다. </li><li> 525 nm의 570에서 543 nm의 방출 수집에 SNARF-1 여기 - - 600 nm의 505에서 405 nm의 방출 수집에 HPTS 여기에 해당하는 첫 번째 단계를 시작합니다. </li><li> 525 nm의 640에서 543 nm의 방출 수집에 SNARF-1 여기 - - 650 nm의 505에서 458 nm의 방출 수집에 HPTS 여기에 해당하는 두 번째 단계를 시작합니다. </li><li> 공 초점 조리개는 기본적으로 175 μm의 설정 및 최적의 조리개를 수동으로 300 μm의에서 설정 될 필요가있다. </li><li> 0.4 μm의 두께의 직렬 수평 광학 절 (800 × 800 픽셀)를 적층하여 이미지를 가져 가라. </li></ul></li></ol> 참고 : 40X 목적은 동시에 여러 개의 세포를 관찰하는 데 유용하고 세포 기관보기에 적합합니다. 그러나, 공간 해상도 및 신호 강도는 베티 아르R은 60X 오일 목적을 사용하여 달성. 또한,이 색소 사이의 여진 또는 발광 크로스 토크는 없다. pH를 감지 프로브의 7. 교정 <ol><li> 교정하기 전에, 이전에 HPTS와 SNARF-1 배양 배양 접시에서 매체를 제거 멸균 PBS로 두 번 세척 한 다음 다섯 교정 솔루션 중 하나 2 ㎖를 추가합니다. </li><li> 공 초점 무대 챔버로 배양 접시를 놓습니다. </li><li> 다음 설정에 따라 시간 경과 이미지 수집을 진행 : <ol><li> 실험에 대한 이미지와 간격 기간의 횟수를 설정합니다. 참고 : 우리는 일반적으로 최대 30 분 동안 이미지 사이에 1 분 간격을 사용합니다. </li><li> 각 이미지의 취득 사이에 닫습니다 셔터를 프로그램. </li></ol></li><li> 스프레드 시트 소프트웨어에 분당 형광 강도를 기록하고 형광 강도가 상승 할 때의 시간을 결정한다. 20 분 - 세포 내 pH는 15 일 이후 일반적으로 안정적이다. </li><li> REPEAT는 나머지 각 교정 솔루션을위한 새로운 배양 접시를 사용하여 7.4 7.1 단계를 반복합니다. </li></ol> 8. 데이터 수집 <ol><li> 교정이 완료되면, 필드 당 15 ~ 20 세포를 포함하는 4-5 무작위로 선택한 분야에서 샘플 데이터를 수집. 두 이미지는 각 필드에 대해 얻을 수 있습니다 : HPTS 라벨 (소포)와 SNARF-1 라벨 (세포질)에 대한 두 번째 하나. </li><li> 배경 형광 (세포 외 영역)는 디지털 두 프로브의 형광 이미지에서 차감됩니다. 각각의 저장된 이미지에서 레이블이 소포 및 세포질은 바이너리 마스크를 사용하여 선택됩니다. 각각의 pH를 감지 프로브를 들어, 형광 강도는 수치 척도 (0-4,095 회색 수준의 규모)에 측정된다. 소기관 또는 세포질 내의 모든 픽셀의 평균 휘도가 계산되고 형광 값의 비율을 계산하는 데 사용된다. </li></ol> 9. 데이터 분석 <ol><li> 다양한 소프트웨어 패키지는 데이터 분석을위한 상업적으로 이용 가능하다. 즉Fluoview 소프트웨어. </li><li> 다음 스프레드 시트, 레코드 값과 형광 비를 계산 HPTS의 경우, R = (F 458 ㎚ / F 405 NM)와, SNARF-1, R = (F 644 ㎚ / F 584 ㎚의 경우 ). </li><li> 같은 그래프 패드 프리즘 과학적 그래프 소프트웨어를 사용하여 데이터를 그린다. 플롯은 산도 (X 축)의 함수로서 형광 비율 (Y 축) 묘사한다. 커브 피팅 비선형 회귀 분석을 사용하여 달성된다. </li><li> 단계 20.7에 기재된 바와 같이 샘플의 형광 농도 모아서 형광 비율을 계산한다. pH 값으로 비율을 변환하는 보정 곡선을 사용합니다. </li></ol>

<ol>
	<li>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=pH+Homeostasis+of+cellular+organelles.">pH Homeostasis of cellular organelles.</a> News Physiol Sci. 17, 1-5 (2002).</li><li>Webb, B. A., Chimenti, M., Jacobson, M. P., Barber, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulated+pH:+a+perfect+storm+for+cancer+progression.">Dysregulated pH: a perfect storm for cancer progression.</a> Nat Rev Cancer. 11, 671-677 (2011).</li><li>Hinton, A., 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=Function+of+a+subunit+isoforms+of+the+V-ATPase+in+pH+homeostasis+and+in+vitro+invasion+of+MDA-MB231+human+breast+cancer+cells.">Function of a subunit isoforms of the V-ATPase in pH homeostasis and in vitro invasion of MDA-MB231 human breast cancer cells.</a> J Biol Chem. 284, 16400-16408 (2009).</li><li>Overly, C. C., Lee, K. D., Berthiaume, E., Hollenbeck, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+intraorganelle+pH+in+the+endosomal-lysosomal+pathway+in+neurons+by+using+ratiometric+imaging+with+pyranine.">Quantitative measurement of intraorganelle pH in the endosomal-lysosomal pathway in neurons by using ratiometric imaging with pyranine.</a> Proc Natl Acad Sci U S A. 92, 3156-3160 (1995).</li><li>Drose, S., Altendorf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bafilomycins+and+concanamycins+as+inhibitors+of+V-ATPases+and+P-ATPases.">Bafilomycins and concanamycins as inhibitors of V-ATPases and P-ATPases.</a> J Exp Biol. 200, 1-8 (1997).</li><li>Pedersen, S. F., King, S. A., Nygaard, E. B., Rigor, R. R., Cala, 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=NHE1+inhibition+by+amiloride-+and+benzoylguanidine-type+compounds.+Inhibitor+binding+loci+deduced+from+chimeras+of+NHE1+homologues+with+endogenous+differences+in+inhibitor+sensitivity.">NHE1 inhibition by amiloride- and benzoylguanidine-type compounds. Inhibitor binding loci deduced from chimeras of NHE1 homologues with endogenous differences in inhibitor sensitivity.</a> J Biol Chem. 282, 19716-19727 (2007).</li><li>Alfonso, A., Cabado, A. G., Vieytes, M. R., Botana, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-pH+crosstalks+in+rat+mast+cells:+cytosolic+alkalinization,+but+not+intracellular+calcium+release,+is+a+sufficient+signal+for+degranulation.">Calcium-pH crosstalks in rat mast cells: cytosolic alkalinization, but not intracellular calcium release, is a sufficient signal for degranulation.</a> Br J Pharmacol. 130, 1809-1816 (2000).</li><li>Han, J., Burgess, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+indicators+for+intracellular+pH.">Fluorescent indicators for intracellular pH.</a> Chem Rev. 110, 2709-2728 (2010).</li><li>Giuliano, K. A., Gillies, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+intracellular+pH+of+BALB/c-3T3+cells+using+the+fluorescence+of+pyranine.">Determination of intracellular pH of BALB/c-3T3 cells using the fluorescence of pyranine.</a> Anal Biochem. 167, 362-371 (1987).</li><li>Willoughby, D., Thomas, R., Schwiening, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+intracellular+pH+changes+on+resting+cytosolic+calcium+in+voltage-clamped+snail+neurones.">The effects of intracellular pH changes on resting cytosolic calcium in voltage-clamped snail neurones.</a> J Physiol. 530, 405-416 (2001).</li></ol>

No conflict of interest declared.

다른 세포 구획의 라이브 세포 이미징 양적 pH 측정은 정확하게 외부의 도전에 대한 세포 반응에서의 pH의 변화를 측정하기 위해 필요합니다. 그러나 남아있는 주요 장애물 중 하나는 쉽게 구체적으로 세포 구획을 대상으로하는 것입니다. 이 점에서, 몇 가지 연구는 일렉트로 또는 마이크로 인젝션 8-10 의해 세포 내로 도입 세포 내 pH를 지표로 HPTS의 사용을 강조했다. 이 기술은 즉 일렉트?...

<table><tbody><tr><td>Solutions for calibration</td><td></td><td></td><td></td></tr><tr><td>Sodium chloride</td><td>Fischer</td><td>L-11621</td><td></td></tr><tr><td>Potassium chloride</td><td>Fischer</td><td>M-12445</td><td></td></tr><tr><td>D-Glucose (dextrose)</td><td>Fischer</td><td>D16-1</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Fischer</td><td>M65-500</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Fischer</td><td>M-1612</td><td></td></tr><tr><td>Deionized water</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Nigericin</td><td>Calbiochem</td><td>481990</td><td>Toxic by inhalation or in contact with skin</td></tr><tr><td colspan="4">Keep protected from the light at 4 &amp;deg;C</td></tr><tr><td>Culture dishes &amp;empty; 35mm</td><td>BD Biosciences</td><td>354456</td><td></td></tr><tr><td>pH-sensing probes</td><td></td><td></td><td></td></tr><tr><td>8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS)</td><td>Life technologies</td><td>H-348</td><td>Keep away from the light at room temperature</td></tr><tr><td>5-(and-6)-Carboxy SNARF&amp;reg;-1, Acetoxymethyl Ester, Acetate</td><td>Life technologies</td><td>C-1271</td><td>Keep away from the light and humidity at -20 &amp;deg;C</td></tr><tr><td>Dimethyl Sufoxide (DMSO)</td><td>Fischer</td><td>BP231-1&amp;nbsp;</td><td>Harmful</td></tr><tr><td>Phosphate buffered saline (PBS) 100X</td><td></td><td></td><td></td></tr><tr><td>Potassium chloride</td><td>Fischer</td><td>M-12445</td><td>20 g</td></tr><tr><td>Sodium phosphate monobasic</td><td>Fischer</td><td>S369-1</td><td>115 g</td></tr><tr><td>Potassium phosphate monobasic</td><td>J.T Baker</td><td>3246-01</td><td>20 g</td></tr><tr><td>Deionized water</td><td>N/A</td><td>N/A</td><td>Bring to 1 L</td></tr><tr><td>Autoclave and adjust pH to 7.4</td><td></td><td></td><td></td></tr><tr><td>Phosphate buffered saline (PBS) 1X</td><td></td><td></td><td></td></tr><tr><td>PBS 100X</td><td>N/A</td><td>N/A</td><td>50 ml</td></tr><tr><td>Sodium chloride</td><td>Fischer</td><td>L-11621</td><td>40.5 g</td></tr><tr><td>Deoinized water</td><td>N/A</td><td>N/A</td><td>Bring to 5 L</td></tr><tr><td>pH modulators</td><td></td><td></td><td></td></tr><tr><td>Bafilomycin A1</td><td>Sigma-Aldrich</td><td>B-1793</td><td></td></tr><tr><td>5-(N-Ethyl-N-isopropyl)amiloride (EIPA)</td><td>Sigma-Aldrich</td><td>A3085</td><td></td></tr><tr><td>Ammonium chloride</td><td>Fischer</td><td>A649-500</td><td></td></tr></tbody></table>

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.

세포 내 엔도 좀 / 리소좀 세포 구획 모두에서의 pH의 동시 측정을 위해, 비율 계량 형광 pH를 감지 SNARF-1 프로브 및 HPTS를 사용 하였다. HPTS가 엔도 솜 / 리소좀 구획의 비율 계량 산도를 측정 할 수있는 반면 SNARF-1은 세포 내 구획으로 제한됩니다. 16 시간 동안 HPTS있는 셀로드는 엔도 좀 / 리소좀 4의 선택적 지역화 할 수 있습니다. pH를 감지 프로브와 함께로드 HT-1080 세포에서 기록 된 형광의 ...

Watch this Scientific Journal Video about Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy at JoVE.com

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

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 ...

simultaneous-ph-measurement-endocytic-cytosolic-compartments-living

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

14.9K 조회수.  Université de Sherbrooke. 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. 

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

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis.

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

전기 화학 및 형광 현미경 검사 법을 사용 하 여 양성자 펌프 막 효소의 연구에 대 한 단일 Liposome 측정

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

연구

생화학

In vitro Monitoring of Extracellular pH in Real-Time

Early accumulation of neutrophils (PMN) is a hallmark of acute intestinal inflammation. This acute inflammation is either resolved or progresses to chronic inflammation. Without efficient PMN clearance at sites of infiltration, PMN can accumulate and contribute to chronic inflammatory conditions, including the intestinal diseases ulcerative colitis (UC) and Crohn's Disease (CD). The pH in the distal colon in individuals with active UC can range between a pH of 5 and 6, whereas healthy individuals maintain colonic pH in the range of 6.8-7.4. Extracellular pH has been shown to influence both intestinal epithelial cells and the infiltrating immune cells. More specifically, extracellular acidosis significantly impacts PMN. At pH below 6.5, there are increases in the production of H2O2, inhibition of apoptosis, and increases in the functional lifespan of PMN. Given the significant presence of PMN and extracellular acidification at sites of inflammation, we developed a novel model that allows for the monitoring of extracellular pH during PMN transepithelial migration in real time. Here, we describe this model and how it can be utilized to measure both the apical and basal pH during PMN trafficking. This model can be utilized to monitor extracellular pH under a wide range of conditions; including, hypoxia, PMN transepithelial migration, and for extended periods of time.

In vitro Monitoring of Extracellular pH in Real-Time

This article represents a useful in vitro assay to measure changes in extracellular pH during neutrophil (PMN) transepithelial migration (TEM)

Early accumulation of neutrophils (PMN) is a hallmark of acute intestinal inflammation. This acute inflammation is either resolved or progresses to ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

비례 형광 현미경으로 Phagosome 산도 측정

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

면역학 및 감염병학

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.

This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

호중구 Phagosome 이미징 및 세포질은 비례의 pH 표시기를 사용하여

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular ...

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, periodontitis, mucosal infections and, most notably, early childhood caries. In all of these conditions, the pH in the biofilm matrix impacts microbe-host interactions and thus the disease progression. The present protocol describes a confocal microscopy-based method to monitor pH dynamics inside cross-kingdom biofilms comprising Candida albicans and Streptococcus mutans. The pH-dependent dual-emission spectrum and the staining properties of the ratiometric probe C-SNARF-4 are exploited to determine drops in pH in extracellular areas of the biofilms. Use of pH ratiometry with the probe requires a meticulous choice of imaging parameters, a thorough calibration of the dye, and careful, threshold-based post-processing of the image data. When used correctly, the technique allows for the rapid assessment of extracellular pH in different areas of a biofilm and thus the monitoring of both horizontal and vertical pH gradients over time. While the use of confocal microscopy limits Z-profiling to thin biofilms of 75 &#181;m or less, the use of pH ratiometry is ideally suited for the noninvasive study of an important virulence factor in cross-kingdom biofilms.

The protocol describes the cultivation of cross-kingdom biofilms consisting of Candida albicans and Streptococcus mutans and presents a confocal microscopy-based method for the monitoring of extracellular pH inside these biofilms.

공초점 현미경을 사용하여 크로스 킹덤 생물막에서 세포 외 pH 모니터링

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, ...

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

Vacuolar and cytosolic pH are highly regulated in yeast cells and occupy a central role in overall pH homeostasis. We describe protocols for ratiometric measurement of pH in vivo using pH-sensitive fluorophores localized to the vacuole or cytosol. Vacuolar pH is measured using BCECF, which localizes to the vacuole in yeast when introduced into cells in its acetoxymethyl ester form. Cytosolic pH is measured with a pH-sensitive GFP expressed under control of a yeast promoter, yeast pHluorin. Methods for measurement of fluorescence ratios in yeast cell suspensions in a fluorimeter are described. Through these protocols, single time point measurements of pH under different conditions or in different yeast mutants have been compared and changes in pH over time have been monitored. These methods have also been adapted to a fluorescence plate reader format for high-throughput experiments. Advantages of ratiometric pH measurements over other approaches currently in use, potential experimental problems and solutions, and prospects for future use of these techniques are also described.

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

Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).

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 ...

생물학

Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy

In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells maintain organismal homeostasis and immunity. Simultaneously, and in contrast to its homeostatic role, it can also drive various pathologies, including cancer and viral infections. Unlike other modes of endocytosis, the tools developed for studying the maturation of macropinosomes remain underdeveloped. Here the protocol describes newly developed tools for studying the redox environment within the lumen of early and maturing macropinosomes. Methodologies for using ratiometric fluorescence microscopy in assessing the pH, production of reactive oxygen species, and the degradative capacity within the lumen of individual macropinosomes in live cells are described. Single organelle measurements offer the advantage of revealing spatiotemporal heterogeneity, which is often lost with population-based approaches. Emphasis is placed on the basic principles of dual fluorophore ratiometric microscopy, including probe selection, instrumentation, calibration, and single-cell versus population-based methods.

We describe protocols for measuring pH, oxidative events, and protein digestion in individual macropinosomes in live cells. An emphasis is placed on dual-fluorophore ratiometric microscopy and the advantages it offers over population-based techniques.

이중 불소공포증 비율 현미경 검사를 사용하여 매크로피노좀의 pH, 레독스 화학 및 분해 능력 측정

In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

신진 효모의 세포 내 이입의 양적, 운동 및 높은 처리량 분석을위한 pHluorin의 응용 프로그램

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

형광 현미경으로 살아있는 세포의 잠재적 인 미토콘드리아의 칼슘과 미토콘드리아 막의 동시 측정

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

pHluorin 태그가 수용체를 활용하여하는 세포 현지화 및 인신 매매를 모니터링하기

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

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

요약

더 많은 비디오 탐색

이 비디오의 챕터

전기 화학 및 형광 현미경 검사 법을 사용 하 여 양성자 펌프 막 효소의 연구에 대 한 단일 Liposome 측정

In vitro Monitoring of Extracellular pH in Real-Time

비례 형광 현미경으로 Phagosome 산도 측정

호중구 Phagosome 이미징 및 세포질은 비례의 pH 표시기를 사용하여

공초점 현미경을 사용하여 크로스 킹덤 생물막에서 세포 외 pH 모니터링

액포와 세포질의 pH 측정

이중 불소공포증 비율 현미경 검사를 사용하여 매크로피노좀의 pH, 레독스 화학 및 분해 능력 측정

신진 효모의 세포 내 이입의 양적, 운동 및 높은 처리량 분석을위한 pHluorin의 응용 프로그램

형광 현미경으로 살아있는 세포의 잠재적 인 미토콘드리아의 칼슘과 미토콘드리아 막의 동시 측정

pHluorin 태그가 수용체를 활용하여하는 세포 현지화 및 인신 매매를 모니터링하기