이 연구는 건강 연구 (CIHR) 운영 보조금 MOP-123419, 자연 과학 및 엔지니어링 연구 위원회의 캐나다 발견 그랜트 418194, 그리고 온타리오 교육부의 연구 및 BH 혁신 초기 연구 수상의 캐나다 학회에 의해 투자 되었다. DGW는 프로토콜의 최적화 및 원고;의 쓰기에, 제시 하는 이미지의 일부를 기여 그는 리버풀 대학에서에서 펌프 못쓰게 교부 금에 의해 투자 되었다. CY는 Vanier 대학원 장학금 및 CIHR MD/PhD 재학에 의해 자금입니다. 자금 지원 기관 연구 설계, 데이터 수집 및 분석, 결정 게시 또는 원고의 준비에 전혀 역할을 했다.

저자는 충돌의 관심을 선언합니다.

이 프로토콜에서 설명 된 방법을 사용 이미징 및 고정 셀 및 라이브 셀 접근을 사용 하 여 동적 efferocytic 프로세스의 정량화. 이러한 방식을 일반적으로 고용된 흐름 cytometry 기반 방법23,24에 여러 가지 이점을 제공합니다. 내부 고정 샘플 얼룩의 사용 속도의 더 강력 하 고 정확한 정량화와 efferocytosis의 범위를 제공 한다-실제로, 많은 교류 cytometry 기반 방...

Apoptosis, 또는 프로그램 된 세포 죽음 대부분 다세포 유기 체에서 발생 하 고 그들의 발달 및 항상성1에 대 한 중요 한 높은 규제 생리 적 과정 이다. 정상 세포 회전율 및 배아 개발에 참여 하 고, 이외에 apoptosis 조직에서 감염 되거나 손상 된 세포의 제거를 가능 하 게 하 고 또한 감염, 염증, 암, 응답 하 고으로 의료 개입에 의해 트리거할 수 있습니다. 방사선 치료 또는 스테로이드1등. Apoptotic 세포 노출 "먹고-나" 다양 한 직업 및 비 직업적인 식 세포에 수용 체에 의해 인식 되는 그들의 세포 표면에 신호 총칭 하 여 "efferocytes"로. 이 수용 체의 참여 efferocytosis2,3로 알려진 프로세스를 통해 efferocyte에 의해 통풍 관 및 apoptotic 세포의 저하를 유도 합니다. Phosphatidylserine은 최고의 특징이 먹고-저 운전 efferocytosis 신호. 그것은 일반적으로 apoptosis을 따라서4셀 표면 phosphatidylserine 노출이 막 비대칭 방해 지질 scramblase 활성화와 원형질 막의 내부 자료에 국한 된다. Phosphatidylserine은 성숙한 세포 등 일부 비 apoptotic 세포의 세포 외 표면에 있고 혈소판 활성화. 그러나,이 세포는 하지 "" 나를 안 먹는, 같은 신호 CD47, 그들의 세포 표면5,,67의 존재로 인해 efferocytosed. 노출된 phosphatidylserine efferocytes에 의해 표현 하는 efferocytic 수용 체의 배열에 의해 인식 된다. Phosphatidylserine에이 수용 체의 바인딩을 직접 또는 opsonins의 원조를 통해 활성화 되 나 efferosome8, 막 도약 공포에 apoptotic 세포의 engulfment를 홍보 하는 신호 통로 9 , 10 , 11 , 12.는 efferosome endosomes를 순차적으로 퓨즈와 리소좀, 분자 기계는 efferosome를 시 어 지 다 하 고 저하는 apoptotic 필요한 제공 셀 화물13,14. 일단 저하, apoptotic 세포 유래 자료는 재활용 endosome에 인신 매매-apoptotic 세포 유래 항 원에 면역 응답을 제한 하 고는 apoptotic 세포13에서 영양분의 복구에 대 한 수는 프로세스 15. Apoptotic 세포;의 장애인된 허가에 efferocytosis 결과에 실패 이러한 uncleared 세포는 결국 보조 괴 사를 받 다. 회 저 성 세포 프로-염증 성 cytosolic 내용, 병원 균, 그리고 autoantigens 따라서 감염, 염증, 자기 면역 질병16,17의 연습장 extracellular 환경으로 풀어. 함께, apoptosis와 efferocytosis 죽어가는 및 죽은 세포의 제거를 촉진 하 고 조직의 항상성 유지에 대 한 허용.
Efferocytosis 기본 분자 메커니즘을 조사 apoptotic 세포 통풍 관의 명확한 정량화를 제공 하는 메서드가 필요 합니다. 이 정량화는 다른 글귀와 달리 endocytosis 및 먹어서18,19, efferocytosis 메커니즘 발생할 수 있습니다 하지 증분 통풍 관의 결과로 그대로 목표 셀의 engulfment에서 사실에 의해 복잡 하 게 efferocyte20apoptotic 세포. 여기에 설명 된 프로토콜 설명 생체 외에서 efferocytosis 분석 결과 내 면의 정확한 묘사를 제공 하는 개별 apoptotic 세포의 비 내 면 부분 대 고정 셀의 다양 한 결합 될 수 있다 및 라이브 셀 현미경에 접근 한다. 전통적인 먹어서 분석 추가 항 체 비 내 면 목표, 우리의 방법으로 레이블 하기 위해 실험의 끝에 phagocytic 대상에 특정 라벨 covalently 연결 biotin21 apoptotic 대상으로 다릅니다 , 22. apoptotic 세포 특정 항 체는이 분석 결과에 사용할 수 있는, biotinylation 접근 어떤 단백질 베어링 대상 표시를 허용 하 고 immunostaining 수행 하는 경우에 이차 항 체 분으로 잠재적인 문제를 피할 수 . 특히, 우리는 셀 추적 염료와 biotin 듀얼 얼룩이 있다 apoptotic Jurkat 세포의 준비 개요. 염료를 추적 셀 apoptotic 세포 유래 자료 efferocytosis 중 추적, 표면 biotinylation의 차별에 대 한 허용 하는 반면 efferocytosed apoptotic 세포의 비 내 면 부분에서 내 면화를 허용 합니다. 우리는 또한 문화 및 준비 J774.2 및 THP-1 셀 라인의 murine 인간과 efferocytes로 사용, M2 세포 monocyte 파생에 대 한 기본 셀 efferocytosis, 및 사용에 대 한 Jurkat 세포의 예를 들어 efferocytic 대상으로 설명합니다. 이러한 방법은 다른 세포 선 또는 1 차 셀, 대상 셀 (예: apoptosis, 괴 사 및 necroptosis), 세포 죽음의 어떤 형태를 겪고 그리고 지질 코팅을 통해 apoptotic 세포 시뮬레이션 미크론 크기의 모방에 쉽게 적용할 수 또는 특정 관심의 efferocytic 수용 체에 ligands와 코팅.
이 프로토콜에서 설명 하는 방법은 필드23,24에 일반적으로 사용 되는 흐름 cytometry 기반 방법 몇 가지 장점이 있습니다. 직접 식 apoptotic 세포 상호 작용, 모두 총 및 비 내 면 apoptotic 세포 물자의 명확한 라벨와 함께 영상으로 efferocytosis의 정량적 측정을 할 수 있다. 또한, pH을 구분 하지 않는 fluorophores의 사용 등 몇 가지 대체 방법을25confounds lysosomal pH에서 FITC와 GFP 형광 억제 혼동 요인 제한 합니다. 마지막으로, 세부 사항에 설명 되지 않은, 하는 동안 이러한 방법을 채택 될 수 있다 transgenes 붙일 표시를 표현 하는 efferocytes를 사용 하 여 또는 후 고정 immunostaining, 신호 분자 활동 및 모니터링의 정량화를 허용 하는 efferocytosis 중 세포 프로세스입니다.

<table>
	<tbody>
		<tr>
			<td>RPMI 1640 Media</td>
			<td>Wisent</td>
			<td>3500-000-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Media</td>
			<td>Wisent</td>
			<td>319-005-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Wisent</td>
			<td>080-150</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Wisent</td>
			<td>311-010-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>18 mm circular glass coverslips #1.5 thickness</td>
			<td>Electron Microscopy Sciences</td>
			<td>72290-08</td>
			<td>Size and shape of coverslip is not critical, but 18 mm fit into the wells of a standard 12-well plate which simplifies cell culture</td>
		</tr>
		<tr>
			<td>Staurosporine</td>
			<td>Cayman Chemical</td>
			<td>81590</td>
			<td>Dissolve in DMSO at 1 mM (1,000x stock solution)</td>
		</tr>
		<tr>
			<td>Annexin V-Alexa 488</td>
			<td>ThermoFisher</td>
			<td>R37174</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ-Link NHS-Biotin</td>
			<td>ThermoFisher</td>
			<td>20217</td>
			<td>Store in a dessicator. Do not prepare a stock solution.</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrace FarRed</td>
			<td>ThermoFisher</td>
			<td>C34572</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrace Orange</td>
			<td>ThermoFisher</td>
			<td>C34851</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoescht 33342</td>
			<td>ThermoFisher</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-Streptavadin</td>
			<td>ThermoFisher</td>
			<td>SA1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Lympholyte-poly cell sepration medium</td>
			<td>Cedarlane Labs</td>
			<td>CL5071</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human M-CSF</td>
			<td>Peprotech</td>
			<td>200-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IL-4</td>
			<td>Peprotech</td>
			<td>300-25</td>
			<td></td>
		</tr>
		<tr>
			<td>J774.2 Macrophage Cell Line</td>
			<td>Sigma-Aldrich</td>
			<td>85011428-1VL</td>
			<td></td>
		</tr>
		<tr>
			<td>THP-1 Human Monocyte Cell Line</td>
			<td>ATCC</td>
			<td>TIB-202</td>
			<td></td>
		</tr>
		<tr>
			<td>Jurkat T Cell Line</td>
			<td>ATCC</td>
			<td>TIB-152</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of efferocytosis by single-cell fluorescence microscopy

Efferocytosis의 규칙을 공부 apoptotic 세포의 통풍 관을 정확 하 게 계량 하 고 efferocytosis를 제어 하는 신호 및 세포 프로세스를 조사 하는 방법을 필요 합니다. 이 정량화 수행 apoptotic 세포 들은 efferocytosed 증분, 따라서 수 정확 하 게 윤곽을 그리 다 잔여 unengulfed 휴대 대 apoptotic 대상의 efferocytosed 부분 사이 방법이 필요로 하기 어려울 수 있습니다. 파편. 여기 설명 된 방법은 이중 라벨 접근 efferocytosis와 efferocytic 용량의 efferocytes 대 식 세포 등의 역학을 정확 하 게 계량을 활용 합니다. Apoptotic 세포의 cytosol apoptotic 세포의 표면 biotinylation 내 면 및 비 내 면 간의 차별화에 대 한 허용 하는 동안 모든 apoptotic 세포 유래 물질의 모니터링을 활성화 하는 셀 추적 염료와 함께 표시 됩니다. apoptotic 세포의 분수 Efferocytes의 efferocytic 용량 라이브 또는 고정 셀의 형광 이미지와 streptavidin 얼룩으로 구분으로 대 내 면된 대상, 바운드의 양을 측정 하 여 결정 됩니다. 이 방법은 cytometry, 즉 비-efferocytosed 대 efferocytosed apoptotic 세포 분수, efferocytic 역학 라이브 셀 현미경으로 측정 하는 능력의 정확한 묘사 같은 방법에 비해 몇 가지 장점이 고 용량을 붙일 레이블 transgenes 표현 하는 세포에 세포 신호의 연구를 수행 합니다. 이 프로토콜에서 설명 하는 방법, efferocytic 활동을 정확 하 게 계량 하 고 efferocytosis 동안 활성 세포 신호 경로 심문 하는 데 사용할 수 있는 유연한 실험적인 접근 방식 기반으로 제공 합니다.

밤새 Jurkat 세포의 apoptosis에 1 µ M staurosporine 결과 문화 > Annexin v (그림 1) 얼룩 확인 될 수 있다 세포의 95%. 비록 staurosporine의 농도 및 staurosporine 치료 기간 각 셀 라인에 대 한 최적화 될 필요가 있을 것 이다 다른 세포 유형,이 실험에 대 한 사용할 수 있습니다. 신뢰할 수 있는 검색 및 efferocytosis의 정량화에 대 한 > 세포의 80%는 efferocytes에 그들을 추가 ?...

Watch this Scientific Journal Video about Quantification of Efferocytosis by Single-cell Fluorescence Microscopy at JoVE.com

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Efferocytosis, phagocytic 제거 apoptotic 세포의 항상성 유지 하는 데 필요한 하 고 수용 체와, engulfment, 인식과 apoptotic 세포의 국제화에 대 한 허용 하는 신호 통로 의해 촉진 된다. 여기, 우리는 efferocytosis의 정량화와 efferocytic 신호 통로의 활동에 대 한 형광 현미경 검사 법 프로토콜을 제시.

단일 세포 형광 현미경 검사 법에 의해 Efferocytosis의 정량화

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

quantification-of-efferocytosis-by-single-cell-fluorescence-microscopy

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JoVE Journal

Immunology and Infection

12.4K 조회수.  University of Western Ontario. Efferocytosis, phagocytic 제거 apoptotic 세포의 항상성 유지 하는 데 필요한 하 고 수용 체와, engulfment, 인식과 apoptotic 세포의 국제화에 대 한 허용 하는 신호 통로 의해 촉진 된다. 여기, 우리는 efferocytosis의 정량화와 efferocytic 신호 통로의 활동에 대 한 형광 현미경 검사 법 프로토콜을 제시.

비디오: Quantification of Efferocytosis by Single-cell 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

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

의 단일 세포 분석 바실러스 subtilis</em형광 현미경 및 유동세포계측법 사용> Biofilms

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

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 ...

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

액포 파열의 단일 세포 측정은 세포 내 병원균에 의해 발생

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

포유 동물 세포와 관련있는 박테리아의 생존을 확인하는 방법에 대한 형광 현미경 방법

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

공 초점 현미경으로 고립 된 폐포 대 식세포의 반대로 곰팡이 활동을 평가

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

공포 성 대 세포질의 정량화<em&gt; 살모넬라</em&gt; 차동 투과성으로하여 기본 대 식세포에서

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

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

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

비례 형광 현미경으로 Phagosome 산도 측정

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

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

개 호중구 세포 외 트랩 릴리스의 정량화를위한 ​​간단한 형광 분석

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.