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. 두 개의 1.5 ml의 튜브에 500 μl를 전송합니다. 5 분 동안 11,000 XG에 원심 분리기. </li><li> 500 μL PBS로 세척을 반복합니다. 자이 모산 비등 냉동 -20 ℃에서 보관, 분주 될 수있다. </li><li> 갓 CO 3 0.1 M 나 2를 준비 500 μL, pH가 9.3로 위 (단계 1.2)에 두 번 모산을 씻으십시오. </li><li> 최종 세척 후 490 μL에 재현 탁. 10 mg을 10 μl를 추가 / FITC 디메틸 sulfoxid에 용해 ㎖로E (DMSO), 소용돌이, 호일로 커버하고, 실온에서 4 시간 동안 교반 품어. </li><li> 처음으로 0.5 ml의 DMSO + 300 μL PBS, 0.5 ml의 DMSO + 450 μL PBS, 다음 PBS 혼자 다음 + 150 μL PBS 0.5 ml의 DMSO와, 상기와 같이 (단계 1.2) 세척, 언 바운드 염료를 제거하십시오. </li><li> 혈구를 사용하여 모산를 계산합니다. 500 μL PBS, 소용돌이에 모산의 1 μl를 추가, 다음은 중앙 그리드 위에 배치 커버 슬립을 충족 혈구 챔버의 가장자리에 10 μl를 추가합니다. 20X 목표를 장착 밝은 필드 현미경의 혈구를 탑재합니다. </li><li> 16 사각형을 포함하는 왼쪽 상단에 초점 손 집계 카운터를 사용하여이 지역의 모든 모산 입자를 계산합니다. 오른쪽 하단에 반복합니다. = 모산 농도 / 2 × 500 × 104 모산 / ㎖ 카운트 : 다음의 방정식을 사용하여 농도를 산출 모산. 표지 모산 냉동 -20 ℃에서 보관, 분주 될 수있다. </li><li> 레이블 모산 위스콘신 Opsonize(100) 희석 : 1 토끼 항 모산 항체 토륨. 소용돌이와는 37 ° C의 물을 욕조에서 1 시간 동안 배양한다. (단계 1.2) 위와 같이 500 μL PBS로 세척 할 것. 참고 : 모산 나중에 분석을 더 어렵게 만들 수 있습니다 덩어리를 형성하는 경향이 초음파 처리는 매우, 특히 옵 소닌 후, 권장합니다. 65 % V / V 쥐 또는 인간 혈청과 옵 소닌은 대안으로 사용될 수있다. </li><li> 단계 1.7에서 1.8 사이에서 설명한대로 혈구 계를 사용 모산을 계산하고, 100 × 106 입자 / ㎖로 희석한다. 이러한 보완 또는 전혀 옵 소닌과 같은 다른 식세포 자극과 옵 소닌은 선택적으로 수행 될 수있다. </li></ol> 2. 라이브 비디오 현미경 <ol><li> 다른 곳에서 38 세부 사항에 설명 된대로 기본 마우스 호중구를 분리합니다. <ol><li> 또한, 모든 탐식 세포 유형을 사용합니다. 로스웰 파크 메모리얼 연구소 (RPMI) 5 % 소 태아 혈청 (V / V), 200 U 페니실린 / streptomyci 보충 1640 배지에서 얼음에 호중구 유지N, 10 × 106 세포 / ml의 농도로 사용 전까지. 다른 세포 유형 1-4 일전 접종 할 수있다. </li></ol></li><li> , 35mm 유리 - 바닥 접시의 중앙에 호중구의 2 × 105 (20 μL)를 첨가 배지의 50 μl를 첨가하여 강하를 확산 및 세포가 부착 할 수 있도록 5 분 동안 37 ℃에서 배양한다. </li><li> 행크의 균형 잡힌 염 용액 (HBSS) 1 ㎖가 특정 MPO 억제제 4- 아미노 히드라 지드 (4- ABH, 10 μM)을 함유하는 37 ℃로 가온 추가. 나트륨 아 지드 (5 mM)을 불특정 MPO 억제제도 이용 될 수 있지만, 최근의 pH 39에서 추가 비특이적 효과를 발휘하기 위해 제안되어왔다. </li><li> 37 ° C 난방 시스템과 40X 오일 목적으로 장착 된 위드 형광 현미경에 접시를 탑재합니다. 세포 및 장비 평형화 있도록 10 분 동안 촬상 않고 세포를 배양한다. </li><li> 명 시야 투과 이미지를 획득 현미경 설정 조정 [PHA자체 또는 440 nm의 또는 490 nm의 여기 및 535 nm의 방출 매 30 초와 가능한 경우 미분 간섭 대비 (DIC)]. </li><li> 너무 광표백 및 광독성 및 실험 질문에 따라 요구되는 것은 피하기 위해 현미경 시스템에있어서의 노출 시간 및 주파수 획득을 조정한다. 이미지 수집을 시작합니다. </li><li> 2 분 후, 10 × 106 (50 μL)를 FITC-모산 접시의 중앙에 추가 옵 소닌. 사용 식세포와 대상의 종류에 따라 세포의 비율 : 대상을 조정합니다. </li><li> 30 분 동안 이미지를 계속합니다. 30 분 후 시간 경과 수집을 중지하고 타이머를 시작합니다. 무대를 이동하고 모든 5 분 이내에, 이미지 다른 phagosomes에보기의 다른 분야에서 10 개 이상의 스냅 샷을 캡처합니다. </li></ol> 3. 교정 및 제어 실험 <ol><li> 실험 일에 (레시피는 표 1에 기재되어있다) 이전에 pH 보정 용액을 제조하고 10 ㎖ 분취 량으로 동결.수조에서 37 ℃로 교정 솔루션과 따뜻하게 녹여. pH 미터와 pH를 확인하고 솔루션의 실제 산도를 기록합니다. </li><li> 이전 이미지 수집에 유리 바닥 접시에 연동 펌프를 장착하고 (제 2) 상기와 같이 라이브 비디오 현미경을 수행합니다. </li><li> 30 분 획득주기의 끝에서, 접시 내의 용액을 제거에 펌프를 켜고 제 교정 액 1ml를 추가 펌프를 멈춘다. </li><li> 신호가 보통 5 분 안정 될 때까지 기다립니다. 각각의 보정 용액으로 반복합니다. <ol><li> 실험 하루에 적어도 하나의 보정을 수행하고, 높은 pH를 시작하여 순차적으로 낮은 작동뿐만 아니라 낮은 산도에서 시작하여 가장 높은쪽으로 순차적으로 보정 작업. </li></ol></li><li> 대조군으로서, 유리 접시 바닥에 900 ㎕의 세포 HBSS HBSS로 희석 된 100 μM NADPH 산화 효소 억제제, 디 페닐 요오드 propionium 100 ㎕ (DPI)를 추가하고,10 분 동안 품어. </li><li> 인수를 시작하고 (단계 2.7) 위와 같이 모산를 추가합니다. 식세포의 15 분 후, HBSS로 희석 (콘카) concanamycin 10 μM V-ATPase의 억제제의 10 μL를 추가하고 추가로 15 분 동안 영상을 계속한다. </li></ol> 4. 분석 <ol><li> 시간 경과 영화와 phagosome 스냅 샷의 분석. 참고 : 다음 지침은 ImageJ에 대한 특정이다. 단계별 설명은 사용되는 소프트웨어 따라 광범위하게 변할 수 있지만,이 섹션에 설명 된 기능은 가장 전문 이미지 분석 소프트웨어로 제공된다. <ol><li> 전문 이미지 분석 소프트웨어 이미지를 연다. 어떤 세포가없는 지역에 사각형 다각형 선택 도구를 사용하여 작은 사각형 투자 수익 (ROI)을 그리기 및 투자 수익 (ROI)에서 플러그인&gt; BG의 뺄셈을 클릭하여 490 채널의 배경을 뺍니다. 그런 다음&gt;을 클릭 편집&gt; 선택하여 440 채널에서 동일한 투자 수익 (ROI)을 업로드 선택과 반복을 복원합니다. </li><li>다음 작업 드롭 다운 메뉴에서 &#39;분할&#39;을 선택, ... 프로세스&gt; 이미지 계산기를 클릭 image1에와 이미지 2 드롭 다운 메뉴에서 해당 이미지를 선택하여 440 채널로 나눈 490 채널의 32 비트 비율의 이미지를 만들 및 32 비트 (플로트) 결과의 체크 박스를 체크. </li><li> &gt; 레인보우 RGB를 이미지&gt; 조회 테이블을 클릭하여 비 호환 색상 코딩 색상 코딩 룩업 테이블을 변경합니다. 임계 값 비율 이미지&gt; 임계 값&gt; 이미지를 클릭하여 0과 무한대 픽셀을 제거 조정하는 .... NaN의 확인란 설정 배경 픽셀이 선택되어 있는지 확인하고, 그 모산은 빨간색으로 표시되도록 스크롤 막대를 조정하고 적용을 클릭합니다. </li><li> 이미지를 클릭하여 멀티 채널 합성으로 시야의 채널이 비 스택 수확기&gt; 컬러&gt; 채널 병합하고 회색 채널 드롭 다운 메뉴의 명 시야 화상과 녹색 채널 드롭 다운 메뉴의 비 화상을 선택하고, 선택체크 상자를 원본 이미지를 유지합니다. 시간 경과 영화 또는 phagosomes 분석 할 수있는 결정하기 위해 스냅 샷을 검색합니다. 밝은 필드 채널이 유용합니다. </li><li> 추가&gt; phagosomes 주위에 타원형 다각형 선택 도구를 사용하여 관심 영역 (ROI)을 그리기 및 분석&gt; 도구&gt; 투자 수익 (ROI) 관리자를 클릭하여 투자 수익 (ROI) 관리자에 추가합니다. 더&gt; 다중 측정 및 슬라이스 체크 상자 당 하나의 행을 선택 해제를 클릭하여 자신의 모산 강도 비를 측정한다. </li><li> 저속 영화, 트랙은 각 시점을 측정하는 M + Ctrl 키를 입력 ROI 드로잉하고 시간의 강도 값을 추적 할 때 필요한 ROI를 이동하여 한 번에 하나씩 phagosomes. 스프레드 시트 소프트웨어에 결과 창에서 측정 값을 복사합니다. 참고 : 독립적 인 실험 15 (실험 × 3 당 5 시간 코스)의 분석은 이상하지만, 더 많거나 적게 관찰 된 변화에 따라 요구 될 수있다. ROI를 저장하는 것은 항상 좋습니다. 일부 소프트웨어 패키지는 허용 영상 정합stration와 로아의 동적 업데이트, 분석의이 부분을 촉진. </li></ol></li><li> 형광의 pH 값으로 변환 <ol><li> 4.1 절에 설명 된대로 교정 시간 경과 영화 phagosomes에 대한 440분의 490의 비율을 측정합니다. </li><li> 스프레드 시트에서, 시간의 경과 비율 값을 플롯하고, 각 pH 보정에 대응하는 시간 기간의 중간에서 5 시간 지점에서 (5-20 사이, 보통) 시야 내의 모든 phagosomes에서 평균 비율 값을 계산 해결책. (또한 그림 5A에서 빨간색 상자를 참조하십시오.) </li><li> 실제 측정의 pH에​​ 대한 이들의 평균 440분의 490 비율의 값을 플롯. 하나의 그래프에 적어도 3 개의 독립적 인 교정을 결합합니다. 일반적으로 S 자형 방정식, 비선형 회귀 (최적 곡선)을 할 수있는 통계 나 수치 소프트웨어 패키지를 사용합니다. 참고 : 예를 들어,도 5b에 사용되는 방정식은 볼츠만 S 자형이었다 [Y = 아래 + ((상위 - 하위)/ (1 + EXP ((V50-X) / 기울기)) Y 값 440분의 490 비율이다], X는 pH 값과 상단, 하단은, V50 및 경사 매개 변수가 소프트웨어에 의해 계산되는가이다. </li><li> pH로 비율 값을 변환 얻어진 방정식을 사용합니다. 참고 : 예를 들어,도 5b에 아 지드 화 나트륨의 존재하에 검량선 얻은 식이다 440분의 490 비 = 1.103 + [(2.89에서 1.103 사이) / (1 ​​+ EXP ((6.993-산도) /0.9291)] . 산도에 대한 해결하면 다음과 같은 식을 제공합니다 : 산도 = 6.993-0.9291 * [LN ((1.178 / (비 - 1.103)) - 1))]. </li></ol></li></ol>

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The authors have nothing to disclose.

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.

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 ...

measuring-phagosome-ph-by-ratiometric-fluorescence-microscopy

Research

JoVE Journal

Immunology and Infection

13.9K 조회수.  University of Geneva. 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. 

비디오: Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

전자 레인지 전송 전자 현미경으로 식물 바이러스 질병의 신속한 진단을 도와

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

연구

면역학 및 감염병학

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

라이브 세포 형광 현미경을 사용하여 박테리아 독소 유도 응답의 시각화

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

라이브 식세포에 Phagolysosome 속생의 연구

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

의 생리적 반응을 측정<em&gt; 초파리</em&gt; 라이브 칼슘 이미징을 사용하여 지질 페로몬에 감각 뉴런

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Ratiometric Imaging of Extracellular pH in Dental Biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 &#181;m thickness, but is limited to the pH range between 4.5 and 7.0.

A pH-sensitive ratiometric dye is used in combination with confocal laser scanning microscopy and digital image analysis to monitor extracellular pH in dental biofilms in real-time.

치과 바이오 필름에 세포 외 pH를 비례 영상

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

&quot;Phagosome 폐쇄 분석은&quot;사용하여 입체적으로 Phagosome 형성을 시각화하는 총 내부 반사 형광 현미경 (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

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 ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of disease or occurrence of serious complications. Ektacytometry uses laser diffraction viscometry to measure the deformability of red blood cells subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress. However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid and deformable red cells. This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability. Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood. In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin concentration and hemoglobin composition of the erythrocytes. In addition to measuring deformability, osmotic gradient ektacytometry provides information about the osmotic fragility and hydration status of erythrocytes. These parameters also reflect the hemoglobin composition of red blood cells from sickle cell patients. Ektacytometry measures deformability in populations of red cells and does not, therefore, provide information on the deformability or mechanical properties of individual erythrocytes. Regardless, the goal of the techniques described herein is to provide a convenient and reliable method for measuring the deformability and cellular heterogeneity of blood. These techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders. Sickle cell anemia is one well-characterized example. Other potential disorders where measurements of red cell deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and the hemolytic anemias due to membrane defects.

Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

Deformability 및 Ektacytometry에 의해 혈액이 질 레드 셀 측정

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

생체 외에서 형광 현미경 검사 법에 의해 개 호 중구 Extracellular 함정 형성: 역동적이 고 양적 분석

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...