우리는 자이스 Confocor 3 장비의 유지 보수 및 셀 측정에 관한 유용한 팁 박사 Vladana Vukojeviç, 분자 의학 센터, 카롤린스카 연구소 감사합니다. 이 연구는, 매그너스 Bergvalls stiftelse 및 Stiftelsen Claes가 Groschinskys의 minnesfond에서 Vetenskapsrådet에서 보조금 (1629 허가 번호 2012-)에 의해 투자되었다.

<ol>
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The authors have nothing to disclose.

FCS이 프로토콜은 면역 세포 (뮤린, 인간 또는 다른 종) 모든 유형의 표면 분자의 분자 동력학 평가를 위해 사용될 수있다. 아래 살아있는 세포의 단일 분자 해상도 FCS 조치 시공간 분자 역학. 분자 밀도뿐만 아니라, 관심있는 단백질의 확산 속도 및 클러스터링 역학은 자기 상관 곡선으로부터 추출 될 수있다. 형광 라벨에 성공 FCS 실험에 대한 중요한 중요하다. 세포막에 관심?...

많은 면역 세포의 기능을 분자 확산과 세포막 내에서의 상호 작용에 의존한다. 생체막들은 복잡하고, 세포막 단백질의 하나에서 병진 확산 속도에 영향을 미칠 수있는 면역 세포의 기능에 중요 할 수있는 많은 요소. 최근 선천성 면역계에 속하는 자연 살해 (NK) 세포, 림프구, NK 세포 활성화 (2)의 상태에 따라 세포막 단백질 두 연구의 차동 확산을 나타내는 것으로 나타났다. 형광 상관 분광법 (FCS)는 생체막 내의 분자 확산 속도를 정량화 할 수있는 기술이다. 그것은의 찬란 고정 볼륨 분자 표지의 평균 확산 속도, 공 초점 현미경의 일반적으로 초점을보고합니다. 그것은 분자 운동에 따라 발생하는 형광의 변동의 측정에 기초정상 상태에서의 시스템. FCS 널리 용액과 지질막 내에 두 형광 염료 및 단백질의 확산을 연구에 사용되었다. 확산 속도에 영향을 미치는 다른 출력 매개 변수는 또한 간접적 방식으로 조사 할 수있다 (예를 들어, 단백질이나 세포막의 분자의 상호 작용의 구조적 변화) (3, 4). FCS는 단일 분자 검출에 대한 가능성을 허용하는 높은 민감도로 인해, 다른 방법에 비하여 우수하다. 그것은 대부분의 단백질 5의 내생 발현 수준에 대한 전형적인 밀리몰 범위로 나노 몰, 분자의 농도에 적합합니다. 다른 대부분의 기술들은 단지 단백질 발현 수준에 대한 상대적 정보를 제공하면서 또한, FCS는 연구 볼륨 내의 단백질의 절대 값의 근사치를 제공 할 수있다. 막은 FLUO 포함 내의 다른 방법은 분자의 확산 속도를 측정(FRAP)을 photobleaching에 후 rescence 복구, 단일 입자 추적 (SPT), 다수의 핀홀 FCS 및 이미지의 상관 관계 방법. FRAP 및 이미지의 상관 관계 방법은 일반적으로 분자 (10)의 절대 수에 대한 정보를 제공하지 않는 앙상블 기법이다. SPT에 비해, FCS의 처리량은 모집단 평균의 특성에 관해서 높다. 레이저 초점 내에 존재하는 분자의 평균 확산 속도가 아니라 단일 분자의 속도보다, 측정 이후 분석은 덜 요구된다. 전문 현미경 (11)을 사용할 수 있습니다하지 않는 한 표준 SPT 라벨은 단일 분자의 식별을 허용하는 것은 매우 낮아야 때문에, SPT는 농도에 대한 정보를 제공 할 수 없습니다. 한편, FCS는 연구중인 분자가 이동되어야합니다. 그것은 단순히 매우 느리게 이동하는 추정 움직이지 분수 또는 분자를 감지하지 않습니다. 분자의 확산 속도 즉긴 획득 시간의 약 1/10 FCS 정확하게 측정 (3) (12)에 표시되지 않을 것보다 초점 내에 상주. 따라서, FCS에 의해 기록 된 확산 계수는에-움직이지-가까운 매우 느린 분수는뿐만 아니라 고려 FRAP와 SPT, 같은 기술에서보고 된 확산 속도보다 빠른 경향이있다. SPT는 것보다 FCS 분자 집단 내에서의 확산 률의 변동에 대한 상세한 설명을 제공 할 것이다. FCS는 흥분 볼륨 내의 시간에 따른 형광 강도의 변동을 정량화한다. 멤브레인의 측정의 경우, 이는 멤브레인의 조명 영역으로 변환한다. 이 논문은 이러한 변동 브라운 확산을 나타내는 분자에 의해 유도되고, 따라서, 및 여진 부피 밖으로 이동된다는 사실을 이용한다. fluctua에 대한 몇 가지 다른 가능한 소스가있다이러한 바인딩 바인딩 해제 전체 세포막의 리간드, 또는 이동의 점멸 또는 형광, 환경 영향의 삼중 상태의 존재와 형광 신호 TIONS. 이러한 추정 에러 소스는 정확한 결과 (12, 13)을 해석하기 위하여 FCS 내의 실험을 설계 할 때 고려 될 필요가있다. 일반적으로 생물 세포막의 측면 확산 속도는 막 단백질 사이 단백질과 세포 골격 사이에 모두 밀집과의 상호 작용에 의한 낮다. 역사적으로, 멤브레인의 FCS의 사용, 따라서 여기 포커스 (14)를 통해 연장 된 전이 시간 동안 탈색을 방지하기 위해 필요한 광 안정 형광체의 부족에 의해 방해되어왔다. 그러나 오늘, 적합한 사진 안정 염료에 대한 많은 옵션이 있습니다. 감지기 및 기타 하드웨어 크게 향상 또한 형광 보호 자전거의 검출을 허용기능과 낮은 밝기의 염료. 여기서, 항체 태그 관심 단백질은 형광 물질로 표지되어 뮤린 차 림프구를 사용하여 FCS의 응용 프로그램을위한 기본 프로토콜이 설명된다. 확산 계수는 분자 밀도를 추출하기 위해 자기 상관 곡선 맞도록 접근법은 또한 도시되어있다. 프로토콜은 쉽게 분자의 확산을 연구하는 기술 된 이전의 경험 면역학 다음되는 것을 목적으로한다. 그러나, 공 초점 현미경의 기본 이해가 예상된다 (참조 (15) 참조 기본적인 이해를 위해). 이 프로토콜은 비교적 용이 다른 현탁 세포 세포주 및 일차 전지 모두 적용될 수있다. 숙련 된 FCS 사용자를 위해 더 정제 된 분석 방법 중 일부는 논의에 기술되어 존재한다.

<table border="0" cellpadding="0" cellspacing="0" width="1206">
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:321px;">MACS NK Cell Isolation Kit mouse II</td>
			<td style="width:367px;">Miltenyi Biotec NordenAB</td>
			<td style="width:181px;">130-096-892</td>
			<td style="width:337px;">Negative selection of NK cells</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Fetal Bovine Serum</td>
			<td style="width:367px;">Sigma-Adlrich</td>
			<td style="width:181px;">F7524</td>
			<td style="width:337px;">Heat inactivated</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Phosphate Buffered Saline</td>
			<td style="width:367px;">-</td>
			<td style="width:181px;">-</td>
			<td style="width:337px;">Made in house&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Roswell Park Memorial Institute medium 1640</td>
			<td style="width:367px;">PAA The Cell Culture Company&#160;</td>
			<td style="width:181px;">E15-848</td>
			<td style="width:337px;">Transparent medium</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Antibody clone 2.4G2</td>
			<td style="width:367px;">Thermo Fischer Scientific</td>
			<td style="width:181px;">553140</td>
			<td style="width:337px;">For blocking Fc-receptors.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Anti-Ly49A antibody&#160;</td>
			<td rowspan="2" style="width:367px;"></td>
			<td rowspan="2" style="width:181px;"></td>
			<td rowspan="2" style="width:337px;">Monoclonal antibody made in house and conjugated in house to Alexa fluor 647</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Clone JR9.318</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Anti-H-2Dd antibody&#160;</td>
			<td rowspan="2" style="width:367px;">BD Pharmingen</td>
			<td rowspan="2" style="width:181px;">558915</td>
			<td rowspan="2" style="width:337px;">Conjugated in house to MFP488</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Clone 34.5.8S</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">MFP488</td>
			<td style="width:367px;">Mobiotech</td>
			<td style="width:181px;">MFP-A2181</td>
			<td style="width:337px;">Fluorescent dye for antibody conjugation.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Poly-L-Lysine</td>
			<td style="width:367px;">Sigma-Aldrich</td>
			<td style="width:181px;">P8920</td>
			<td style="width:337px;">Diluted in distilled water (1.10)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:321px;">Poly-L-Lysine (20 kDa) grafted with polyethylene glycol (2 kDa)</td>
			<td style="width:367px;">SuSoS AG</td>
			<td style="width:181px;">PLL(20)-g[3.5]-PEG(2)</td>
			<td style="width:337px;">Diluted in PBS (pH 7.4) to 0.5 mg/ml.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:321px;">Rhodamine 110 chloride</td>
			<td style="width:367px;">Sigma-Aldrich</td>
			<td style="width:181px;">432202</td>
			<td style="width:337px;">Known diffusion coefficient: 3.3 &#215; 10&#8722;10 m2/s 19</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:321px;">Alexa fluor 647</td>
			<td style="width:367px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:181px;">A20006</td>
			<td style="width:337px;">Known diffusion coefficient: 4.4 &#215; 10&#8722;10 m2/s 20</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Confocal microscope&#160;</td>
			<td style="width:367px;">Zeiss</td>
			<td style="width:181px;">LSM510</td>
			<td style="width:337px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Software: Confocor 3</td>
			<td style="width:367px;">Zeiss</td>
			<td style="width:181px;"></td>
			<td style="width:337px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Software: Matlab with curve fitting toolbox</td>
			<td style="width:367px;">Matlab</td>
			<td style="width:181px;">Version R2013b</td>
			<td style="width:337px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Nunc Lab-Tek Chambered Coverglass</td>
			<td style="width:367px;">Thermo-scientific&#160;</td>
			<td style="width:181px;">155411</td>
			<td style="width:337px;">8 wells, 1.0 borosilicate bottom</td>
		</tr>
	</tbody>
</table>

molecular diffusion in plasma membranes of primary lymphocytes measured by fluorescence correlation spectroscopy

형광 상관 분광법 (FCS)은 높은 시공간 해상도 생체막 내 분자의 확산을 연구하는 강력한 기술이다. FCS는 세포막에서 형광 표지 된 분자의 분자 확산 계수 및 농도를 정량화 할 수있다. 이 기술은 (실제 측정 시간 동안의 결과에 영향을 미치는 일없이, IE) 정상 상태가 면역 세포의 세포막 분자의 분자 확산 특성을 조사하는 기능을 갖는다. FCS는 대부분의 내인성 단백질의 전형적인 수준으로 발현되는 단백질의 확산을 연구하는데 적합하다. 여기서, 차 림프구의 세포막 단백질의 확산 속도를 결정하기위한 간단하고 강력한 방법이 설명된다. 항체 염색 생균 및 수집 한 후 통상적으로 발생 관측에 대한 측정을 수행하는 효과적인 방법을 설명한다. 최근 발전광 안정 형광 염료의 개발에 광범위 측정 동안 표백하지 염료 적절한 관심 항체 컨쥬 게이트가 이용 될 수있다. 또한이 세포막에 발현 된 단백질의 공통 특징이다 천천히 확산 엔티티의 검출을 허용한다. 생성 된 자기 상관 곡선에서 분자 농도와 확산 매개 변수를 추출하는 분석 절차가 강조 표시됩니다. 요약하면, FCS 측정을위한 기본 프로토콜이 제공된다; 그것은 공 초점 현미경의 이해되지만, 분자 확산 속도와 같은 동적 매개 변수를 측정하기위한 기술의 다른 이전의 경험과 면역 학자 다음에 할 수 있습니다.

전형적인 결과는 막 단백질 400 밀리 초 내지 10 밀리 초 범위의 통과 시간과 자기 상관 곡선을 생성한다. 분자의 수는 내생 적으로 발현 된 단백질에 대한 약 200 μm의 2 당 0.5 사이에 다를 수 있습니다. 노출 당 비용이 예상보다 낮지 것을주의 깊게 확인합니다. 이것은 백그라운드 신호의 영향이 있다는 것을 의미 할 수있다. 엄지 손가락의 규칙으로 분석을 받아 들?...

Watch this Scientific Journal Video about Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy at JoVE.com

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

형광 상관 분광학에 의해 측정 된 차 림프구의 플라즈마 막 분자 확산

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

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10.8K 조회수.  Karolinska Institutet. A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

비디오: Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

세포 독성 T Lymphocytes에 의해 베타 세포 죽음의 중재를 평가하는 방법

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

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piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

기본 인간 T 세포 piggyBac Transposon 시스템 변경

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

PNA-FISH에 의해 마우스 B 림프구에서 염색체 이상을 신속하게 분석

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

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 ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

구조화 조명 현미경 데이터의 시공간적 이미지 상관 분광학에 의해 정량화 T 세포에서 대뇌 피질 굴지 흐름

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

HIV-1 감염 라이브 차 인간 대 식세포에서 측정 Phagosome 마이그레이션 및 속도

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

기자 마우스를 사용하여 IL-22 발현 림프구의 시각화

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

마우스 작은 장의 면역 시스템에서 세포를 분리

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

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 ...