Name: measuring phagosome ph by ratiometric fluorescence microscopy
Uploaded: 2015-12-07T14:00:00
Description: Watch this Scientific Journal Video about Measuring Phagosome pH by Ratiometric Fluorescence Microscopy at JoVE.com

The authors are financially supported by the Swiss National Science Foundation through an operating grant N&#176; 31003A-149566 (to N.D.), and The Sir Jules Thorn Charitable Overseas Trust through a Young Investigator Subsidy (to P.N.).

윤리 선언문 : 모든 동물의 조작이 제네바 대학의 동물 연구위원회의 지침을 엄격히 준수 하였다. 식세포 대상 1. 준비 <ol><li> 10 ml의 멸균 인산 완충 식염수 (PBS)에 건조 모산 20 mg을 추가합니다. 10 분간 끓는 수욕에서 와류 및 열. 5 분 2,000 XG에 시원하고 원심 분리기. </li><li> 물 목욕 초음파기에서 10 분 동안 상층 액 1 ml의 PBS에 재현 탁하고 초음파 처리를 제거합니다. ...

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더 많은 시간이 목표물에 커플 링하여 pH 민감성 염료의 사용과 유사한 방법을 적용하지만 phagosomes의 모집단의 평균 pH는 예컨대 분광 측정과 같은 다른 방법 FACS,보다 많이 소요되었지만, 현미경은 몇 가지 장점을 제공한다. 우선 내부와 외부가 내면화 구속,하지만 것입니다, 입자는 쉽게 각각 소멸 또는 외부 입자에 라벨을, 같은 트리 판 블루 또는 항체와 같은 다른 화학 물질을 추가 할 필요없이...

Phagocytosis, the process through which innate immune cells engulf large particles, evolved from the eating mechanism of single-celled organisms, and involves binding to a target, enveloping it with a membrane and pinching the membrane off to form a vacuole within the cytosol called a phagosome. While the phagosomal membrane is derived from the plasma membrane, active protein and lipid sorting, as well as fusion with endomembranes during phagosome formation, transform the phagosome into a distinct organelle within the cell with degradative properties that allow the killing, neutralization and breakdown of the ingested material1-3. This process, called phagosomal maturation, relies on the delivery of a host of proteolytic and microbicidal enzymes, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase which transfers electrons into phagosomes producing the strong oxidant O2- and its derivative reactive oxygen species (ROS) 2,4. 
The luminal pH of phagosomes has a profound influence on several events required for phagosome function. First, pH influences trafficking of endomembranes in general, as pH-dependent conformational changes of transmembrane trafficking regulators alters the recruitment of trafficking determinants such as Arfs, Rabs and vesicular coat-proteins, which in turn define which vesicles may fuse with phagosomes 5-8. Second, the ionic composition of the phagosomal lumen is also transformed as phagosomes mature, and some ion transporters, such as the Na+/H+ exchanger or ClC family Cl-/H+ antiporters, which promote phagocytic function, rely on H+ translocation 9,10. Similarly, ROS production is intimately linked with phagosomal pH. ROS and its toxic oxidant byproducts have long been recognized as crucial for phagosomal killing in neutrophils 4,11,12, and have been shown to play critical roles in other phagocytes including macrophages, dendritic cells (DCs) and amoeba 13-16. The NADPH oxidase is an electrogenic enzyme that releases H+ in the cytosol as NADPH is consumed, and that requires the simultaneous transfer of H+ through companion HVCN1 channels alongside the transported electrons into the phagosomal lumen, in order to alleviate the massive depolarization that would otherwise lead to self-inhibition of the enzyme 17-21. Finally, several proteolytic enzymes have optimal activity at different pH, so time-dependent phagosomal pH changes can influence which enzymes are active and when. The importance of phagosomal pH is highlighted by organisms such as Mycobacterium tuberculosis, Franciscella tularensis and Salmonella typherium that subvert phagocytic killing at least in part by altering phagosomal pH 22-24.
In mammals the main phagocytes are neutrophils, macrophages and dendritic cells, and depending on cell type, time-dependent phagosomal pH changes can vary widely, and appear to play different roles. In macrophages a strong and rapid acidification mediated by the ATP-dependent proton pump vacuolar ATPase (V-ATPase) is one of the key factors mediating killing 25-27, resembling the mechanisms present in amoeba that use phagocytosis as an eating mechanism 28. In these cells activation of acidic proteases is thought to play a key role. In contrast, neutrophil killing relies more on ROS as well as HOCl produced by myeloperoxidase (MPO)11, and the pH remains neutral or alkaline during the first 30 min acidifying only later 29,30. Neutral pH has been suggested to favor the activity of oxidative proteases such as certain cathepsins. In DCs phagosomal pH is controversial, with some reporting acidification and others neutral or alkaline pH 31,32, but ROS and pH may profoundly influence the ability of these cells to present antigens to T cells, one of their main functions 33. 
Importantly, hormones, chemokines and cytokines may produce signaling events that induce maturation and changes in phagocyte behavior, and in turn influence phagosomal pH 34,35. Similarly, drugs, for example the antimalarial chloroquine, which is also considered for anti-cancer therapies 36, may affect phagosomal pH and therefore immune outcomes. Thus, a variety of cell biologists, immunologists, microbiologists and drug developers may be interested in measuring phagosomal pH when seeking to understand the mechanisms underlying the effects of a particular genetic disruption, bioactive compound or microorganism on innate and adaptive immune responses. 
Here we describe a method for measuring phagosomal pH in neutrophils that allowed us to show the importance of the HVCN1 channel in regulating neutrophil phagosomal pH 19. The method is based on the ratiometric property of fluorescein isothiocyanate (FITC) whose fluorescence emission at 535 nm is pH sensitive when excited at 490 nm but not 440 nm 37. When this dye is chemically coupled to a target, in this case zymosan, it can be followed using wide-field fluorescence microscopy, where cells are imaged as they phagocytose, and phagosomal pH changes are measured in real time as the phagosome matures. The actual pH is then gleaned by performing a calibration experiment where cells that have phagocytosed are exposed to solutions of different pH that contain the ionophores nigericin and monensin, that allow the rapid equilibration of the pH within phagosomes with the external solution. Ratio values are then compared to the known pH of solutions, a calibration curve is constructed by nonlinear regression and the resulting equation used to calculate pH from the ratio value.

<table>
	<tbody>
		<tr>
			<td>Zymosan A powder</td>
			<td>Sigma-Aldrich</td>
			<td>Z4250</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate</td>
			<td>Sigma-Aldrich</td>
			<td>F7250</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Anti-zymosan antibody (Zymosan A Bioparticles opsonizing reagent)</td>
			<td>Life Technologies</td>
			<td>Z2850</td>
			<td>Sigma-Aldrich O6637 is an equivalent product. Alternatively 25% serum can be used as an opsonizing reagent.</td>
		</tr>
		<tr>
			<td>4-Aminobenzoic hydrazide (4-ABH)</td>
			<td>Santa Cruz</td>
			<td>sc-204107</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Diphenyleneiodonium chloride (DPI)</td>
			<td>Sigma-Aldrich</td>
			<td>D2926</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Concanamycin A (ConcA)</td>
			<td>Sigma-Aldrich</td>
			<td>27689</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Nigericin</td>
			<td>Sigma-Aldrich</td>
			<td>N7143</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Monensin</td>
			<td>Enzo</td>
			<td>ALX-380-026-G001</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Life Technologies</td>
			<td>14200-075</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Hank&#39;s balance salt solution</td>
			<td>Life Technologies</td>
			<td>14025092</td>
			<td>Ringer&#39;s balanced salt solution or other clear physiological buffers may be substituted.</td>
		</tr>
		<tr>
			<td>Sodium carbonate (Na2CO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S7795</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>2-(N-Morpholino)ethanesulfonic acid (MES)</td>
			<td>Sigma-Aldrich</td>
			<td>M3671&#160;</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>N-Methyl-D-glucamine (NMDG)</td>
			<td>Sigma-Aldrich</td>
			<td>M2004&#160;</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid (EGTA)</td>
			<td>Sigma-Aldrich</td>
			<td>3777</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>&#160;Tris(hydroxymethyl)aminomethane (Tris)</td>
			<td>Sigma-Aldrich</td>
			<td>T1503&#160;</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Magnesium chloride (MgCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Absolute Ethanol (EtOH)</td>
			<td>Sigma-Aldrich</td>
			<td>2860</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Glass-bottom 35 mm petri dishes (Fluorodish)</td>
			<td>World Precision Instruments</td>
			<td>FD35-100</td>
			<td>Ibidi &#181;-clear dishes or coverslips with appropriate imaging chambers may be sustituted</td>
		</tr>
		<tr>
			<td>Sonicating water bath</td>
			<td>O. Kleiner AG</td>
			<td></td>
			<td>A sonicator may be used instead, various instrument providers exist</td>
		</tr>
		<tr>
			<td>Heamocytometer</td>
			<td>Marienfeld GmbH</td>
			<td></td>
			<td>Various instrument providers exist</td>
		</tr>
		<tr>
			<td>Widefield live imaging microscope</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td>Various instrument providers exist, but the microscope must be able to image 440/535 and 490/535 excitation/emission respective. Spinning disk confocal set-ups with brightfield capabilities may substituted, but zymosan tend to go out of focus more often.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Peristaltic pump (Dynamax RP-1)</td>
			<td>Rainin</td>
			<td></td>
			<td>Various instrument providers exist</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Schott Gerate GmbH</td>
			<td></td>
			<td>Various instrument providers exist</td>
		</tr>
		<tr>
			<td>Manual Counter</td>
			<td>Milian SA</td>
			<td></td>
			<td>Various instrument providers exist</td>
		</tr>
	</tbody>
</table>

measuring phagosome ph by ratiometric fluorescence microscopy

식균 작용은 선천성 면역 세포를 죽이거나 무력화 섭취 자료를, 또는 항원으로 제시하고 적응 면역 반응을 시작하기 위해 박테리아, 세포 사멸 세포 또는 다른 이물질을 삼켜 통해 기본적인 과정이다. phagosomes의 pH는 endomembranes 및 단백질 분해 효소, 식세포 공포가 degradative 세포 기관으로 성숙 할 수 있도록 이벤트의 활성화와 분열 또는 융합을 조절하는 중요한 매개 변수입니다. 또한, H +의 전위의 다른 숙주 조직으로 효율적으로 사멸에 필수적인 시그널링되어 반응성 산소 종 (ROS), 높은 수준의 생산을 위해 필요하다. 많은 세포 내 병원균, phagosomal 산성화를 제한 phagosome 생물학에서의 pH의 중요성을 강조 식세포 살해를 파괴. 여기에 우리가 형광 이소 티오 시아 네이트 (FITC)를 사용하여 호중구에 phagosomal의 산도를 측정하는 비율 계량 방법을 설명은 식세포의 TARG으로 모산를 - 표지ETS, 라이브 셀 이미징. 분석은 490 nm에서 여기 때 단일 염료, 오히려 절대 형광보다, pH에 의존하는 비율의 정량화를 허용, 440 nm에서 흥분하지 않을 경우 산성 pH에 의해 급냉 FITC의 형광 특성을 기반으로합니다. 현장 염료 교정 및 실제 pH 값에 대한 비율의 변환에 수행하기위한 세부적인 프로토콜도 제공됩니다. 그들은 같은 표백 등의 교란에 덜 민감로 단일 염료 비율 계량 방법은 일반적으로 단일 파장 또는 이중 염료 의사 비율 계량 프로토콜 우수 고려, 측정 된 신호를 왜곡 변화, 레이저 변화, 고르지 라벨, 초점을 맞 춥니 다. 이 방법은 용이하게 다른 포식 세포 유형에서의 pH를 측정하기 위해 수정 될 수 있으며, 자이 모산 불활성 비드에서 살아있는 미생물, 기타 아민 - 함유 입자에 의해 대체 될 수있다. 마지막으로,이 방법은 다양한 범위의 pH 또는 다른 phagosom 민감한 다른 형광 물질을 사용하도록 구성 될 수있다알 활동, 그것은 phagosomes의 기능 이미징을위한 일반화 된 프로토콜 만들기.

다음 실험에 대한 대표적인 결과 어디에 야생형 또는 골수에서 분리 한 기본 마우스 호중구의 phagosomal 산도 Hvcn1 - / - 마우스를 비교 하​​였다. 성공적인 실험을 위해, 그것은 나중에 이미지 분석 동안 세그먼트에 더 어려울 것이다 너무 많은 phagosomes을 회피하면서, 저속 동영상의 전체 지속 기간 동안 시야 내에 충분히 phagosomes를 획득하는 것이 중요하다. 1은 도표</s...

Watch this Scientific Journal Video about Measuring Phagosome pH by Ratiometric Fluorescence Microscopy at JoVE.com

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

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.

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