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>
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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 border="0" cellpadding="0" cellspacing="0" width="872">
	<tbody>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">Solutions for calibration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Sodium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">L-11621</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Potassium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M-12445</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">D-Glucose (dextrose)</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">D16-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Magnesium sulfate</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M65-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">HEPES</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Calcium chloride dihydrate</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M-1612</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Deionized water</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Nigericin</td>
			<td style="width:136px;">Calbiochem</td>
			<td align="right" style="width:119px;">481990</td>
			<td>Toxic by inhalation or in contact with skin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td style="width:315px;">Keep protected from the light at 4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Culture dishes &#8709; 35mm</td>
			<td style="width:136px;">BD Biosciences</td>
			<td align="right" style="width:119px;">354456</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">pH-sensing probes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS)</td>
			<td style="width:136px;">Life technologies</td>
			<td style="width:119px;">H-348</td>
			<td>Keep away from the light at room temperature</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">5-(and-6)-Carboxy SNARF&#174;-1, Acetoxymethyl Ester, Acetate</td>
			<td style="width:136px;">Life technologies</td>
			<td style="width:119px;">C-1271</td>
			<td>Keep away from the light and humidity at -20&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Dimethyl Sufoxide (DMSO)</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">BP231-1&#160;</td>
			<td>Harmful</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">Phosphate buffered saline (PBS) 100X</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Potassium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M-12445</td>
			<td>20 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Sodium phosphate monobasic</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">S369-1</td>
			<td>115 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Potassium phosphate monobasic</td>
			<td style="width:136px;">J.T Baker</td>
			<td style="width:119px;">3246-01</td>
			<td>20 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Deionized water</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>Bring to 1 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Autoclave and adjust pH to 7.4</td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">Phosphate buffered saline (PBS) 1X</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">PBS 100X</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>50 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Sodium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">L-11621</td>
			<td>40,5 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Deoinized water</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>Bring to 5 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">pH modulators</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">&#160;Bafilomycin A1</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:119px;">B-1793</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-(N-Ethyl-N-isopropyl)amiloride (EIPA)</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:119px;">A3085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Ammonium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">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

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

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

생활의 이미징 셀 모양 변경 Drosophila 태아

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

연구

생물학

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

라이브 세포의 저장소로 작동하는 칼슘 항목의 형광 기반의 측정 : 교양 암 세포에서 골격근 섬유에

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

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 ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

4D 공 초점 현미경을 사용하여 제브라 피쉬의 두개 안면 형태 형성 분석

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

공 초점 및 다중 광자 현미경을 사용하여 다섯 형광 단백질에 의해 표시됨 조혈 줄기 및 전구 세포의 생체 내 클론 추적에서

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

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 ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

고해상도 시간 경과 이미징 및 생활 인간 제대 정맥 내피 세포의 미세 소관 역학의 자동 분석

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

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+ ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

동적 세포 돌출부에서 소프트웨어를 이용한 단백질 농도 추적을위한 그래픽 사용자 인터페이스

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis. N&#38;B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&#38;B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&#38;B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&#38;B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis.

총 내부 반사 형광 현미경 검사법에 의해 살아있는 세포에 있는 세포 표면 수용체의 올리고머화 역학 은 수 및 밝기 분석과 결합됩니다

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...