由加拿大健康研究，白血病和淋巴瘤协会加拿大和加拿大淋巴瘤基金会机构的拨款支持。

准备试剂注：所有PBS含有的Ca 2 +和Mg 2 + <ol><li>检测试剂 20μM7 - 氨基-4 - 氯甲基香豆素，T - BOC -亮氨酸，蛋氨酸酰胺（BOC - LM - CMAC）添加至室温，PBS。每个细胞悬液，将需要2ML的检测试剂。 </li><li> 1％多聚甲醛（定格） 稀4％多聚甲醛在室温PBS原液。每个流式细胞仪管分装1ml含1％多聚甲醛。流式细胞仪三管为每个实验条件的要求（如32Dkit，32Dkit + PD150606，32Dkit + PD98059需要9流式细胞仪管） </li><li> PD150606（蛋白酶抑制剂） 重组在DMSO PD150606到终浓度为100mm。保护此试剂和所有与本实验的持续时间从光线处理的细胞。 </li><li> PD98059（MEK1抑制剂） 重组在DMSO终浓度的PD98059为50mm。 </li></ol>准备细胞<ol><li> × 10 5至1 × 10 6细胞每毫升OPTI - MEM媒体与5％胎牛血清，WEHI - 3上清液作为源（或任何其他来源的IL - 3，IL - 3补充，增长5 32Dkit细胞作为重组蛋白），0.1％，2 - 巯基乙醇和抗生素。 </li><li>收集到三个15ML猎鹰的管（4 × 10 6细胞每管）12 × 10 6 32Dkit的细胞。 </li><li> 5分钟沉淀细胞在1000 rpm，在15 ° C。 </li><li>吸出上清液。 2ML房间温度PBS重悬细胞。 </li><li>治疗50μMPD150606细胞悬液。确保样本覆盖猎鹰管铝箔避光。涡孵育一段时间后二十到三十分钟，在室温下在黑暗中。 </li><li>治疗20μM的PD98059的第二个细胞悬液。板在一个35mm的组织培养皿中孵化，并为2个小时的细胞在37 ° C。 </li></ol>在固定细胞的钙蛋白酶含量<ol><li>虽然样本PD150606孵化，开始对未经处理的样品的蛋白酶检测。从每个细胞悬液加入2ML检测试剂开始。立即加入1ml到先前准备的流式细胞仪管的细胞悬液/检测试剂混合，用1％多聚甲醛（0分钟的时间点）。 </li><li>与检测试剂为5分钟，在室温下孵育细胞悬液。然后加入细胞悬液1ml到一个流式细胞仪管，用1ml 1％多聚甲醛。 </li><li>继续孵育检测混合细胞悬浮液在室温下额外的5分钟。然后加入细胞悬液1ml到一个流式细胞仪管，用1ml 1％多聚甲醛。 </li><li>重复32Dkit已与抑制剂治疗的细胞蛋白酶检测。 </li><li>修复细胞在黑暗中过夜，在4 ° C。 </li><li>第二天沉淀在5分钟1800转的细胞在15 ° C。 </li><li>吸出上清液。在750uL PBS重悬细胞。置于冰上的细胞，并保持在黑暗中。 </li></ol>固定细胞获取数据<ol><li>分析使用一个LSR II（BD生物科学）的细胞。过滤器设置的光波Xcyte（UV）激光，激发线355纳米和激光功率25MW，底物和产物分别为405和450 nm的排放。底物和产物的带通滤波器二十○分之四百〇五和五十分之四百五十零。为底物和产物的长传过滤器405和450。 PMT电压设定在350-375和325-350为底物的产品。 </li><li>设置一个BD FACSDiva带有以下参数的实验：前向散射;侧散射;印支1蓝（日志）;印支1紫（日志）。在工作​​表上设置了以下图表：前向散射对侧向散射对活细胞的门（图1a），​​印度支那1，印度，印支1紫罗兰直方图1蓝与印支1紫蓝色直方图散点图点图。 </li><li>校准与AlignFlow和AlignFlow LSR II PLUS（Invitrogen）的荧光珠。光电倍增管电压应调整放置在每次实验前，相同的信道珠荧光直方图的高峰期。 </li><li>使用32Dkit细胞在0分钟的时间点开始采集数据。调整必要的参数电压。获取和记录10000个样本事件。 </li><li>每个样品重复采集，每管之间的流式细胞仪的缓冲区冲洗机。 </li><li>导出的数据。确保LSR II是根据研究所的指引清洗。 </li></ol>在活细胞中的钙蛋白酶含量<ol><li>上述制备细胞悬液，没有PD150606。的LSR II带来的样品，并设置过滤器和参数。成立上述工作表。包括一个大型的散点图显示印支1蓝与时间。设置方案，以获得最大的NUM事件的BER（没有停止门）。 </li><li>添加50μMPD150606一个32Dkit细胞的细胞悬液。在黑暗中保持样品在室温下孵育20-30分钟。 </li><li>开始获取32Dkit细胞悬液没有PD150606数据。使用低流速每秒200-300事件。 30秒到1分钟后，从机器中取出管。 20μMBOC - LM - CMAC的细胞。涡简要然后继续采集数据。 </li><li> 10-20分钟，或直至蛋白酶活性高原获取数据。 </li><li>与PD150606治疗的32Dkit细胞，重复步骤3和4。 </li><li>导出的数据。确保LSR II是根据研究所的指引清洗。 </li></ol>钙蛋白酶含量的复合（混）的细胞群 *本实验将确定32Dkit收获了从小鼠脾细胞悬液细胞。 <ol><li>收获从每个鼠标的脾脏。裂解红细胞与NH 4氯在室温下10分钟。 </li><li>准备在2ML PBS单细胞悬液。 </li><li>一分为二的每一个细胞悬液。治疗50μMPD150606 1细胞悬液，在室温下在黑暗中为20-30分钟。 </li><li>按照上述固定细胞中的钙蛋白酶含量（步骤1-6） </li><li>吸出上清液。在500μLPBS清洗细胞。吸出上清液。 </li><li> 2％BSA的细胞重悬于200μL染色缓冲。 15分钟在室温下孵育。 </li><li>准备的主要抗体（FITC标记的抗小鼠CD117）。应使用该抗体染色缓冲液在1:200稀释。确保抗体是被蒙在鼓里。 </li><li>加入200μL第一抗体的细胞。孵育30分钟，在室温下在黑暗中。 </li><li>添加400μLPBS增加流式细胞仪的体积。置于冰上的细胞，并保持在黑暗中。 </li><li>按照固定细胞获取数据。加入FITC标记的参数，包括为FITC标记的工作表上的直方图。 </li></ol>分析和代表性的结果<ol><li>使用FlowJo（Treestar）分析数据。门上的活细胞的基础上正向和侧向散射剖面（图1a）。确定印度蓝门（图1b）从活细胞的平均荧光。 </li><li>一）为混合细胞群<ul><li>使用柱状图和点图FITC荧光识别32Dkit人口</li><li>印度蓝确定的32Dkit人口平均荧光</li></ul></li><li>使用Microsoft Excel图表的平均荧光（图2a）。计算之间的5分钟和10分钟线的斜率。图的斜率为条形图，以确定细胞中的蛋白酶活性（图2b）。 </li></ol><img src="/files/ftp_upload/2050/2050fig1a.jpg" alt="图1a" /> 图1a确定前部和侧散射轮廓为基础的活细胞。 <img src="/files/ftp_upload/2050/2050fig1b.jpg" alt="图1b" /> 图1b。平均超过10分钟的时间当然的荧光变化。 <img src="/files/ftp_upload/2050/2050fig2a.jpg" alt="图2a" /> 图2a。代表图的平均荧光。 <img src="/files/ftp_upload/2050/2050fig2b.jpg" alt="图2b" /> 图2b。钙蛋白酶活性抑制剂的32Dkit细胞。

<ol>
	<li>Goll, D. E., Thompson, V. F., Li, H., Wei, W., Cong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Calpain System. Physiol. Rev. 83, 731-801 (2003).</li><li>Niapour, M., Berger, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+Cytometric+Measurement+of+Calpain+Activity+in+Living+Cells.">Flow Cytometric Measurement of Calpain Activity in Living Cells.</a> Cytometry A. 71A, 475-485 (2007).</li><li>Leloup, L., Daury, L., Maz&#233;res, G., Cottin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+the+ERK/MAP+kinase+signalling+pathway+in+milli-calpain+activation+and+myogenic+cell+migration.">Involvement of the ERK/MAP kinase signalling pathway in milli-calpain activation and myogenic cell migration.</a> The International Journal of Biochemistry and Cell Biology. 39, 1177-1189 (2007).</li></ol>

在固定和生活32Dkit细胞的钙蛋白酶活动已确定在这个视频协议。导致完全抑制蛋白酶的活性，在细胞治疗的细胞株的蛋白酶抑制剂PD150606。此外，治疗与MEK1抑制剂PD98059完全抑制蛋白酶的活性，在细胞内。 ERK的是主要激活的钙蛋白酶2 3。这些结果表明，钙蛋白酶2的活动主要是在32Dkit细胞。在我们的实验室目前的工作是调查蛋白酶的活性增加造成的后果和蛋白酶- 1 - 2蛋白酶在白血病的贡献?...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>PBS BOC-LM-CMAC 4% paraformaldehyde</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td> Dissolve 4g paraformaldehyde in 100mL PBS. In fume hood, heat sample with stirring until clear. Let cool then store at 4&deg;C</td>
</tr>
<tr>
<td>PD150606</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>Use freshly reconstituted PD150606 for maximal calpain inhibition.</td>
</tr>
<tr>
<td>PD98059</td>
<td>&nbsp;</td>
<td>Cell Signaling Technology</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>AlignFlow, AlignFlow plus fluorescent beads FACS tubes</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>LSR II</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>&nbsp;</td>
<td> Supernatant of WEHI-3 cells is used as a source of IL-3 in these experiments. Each collection of WEHI-3 supernatant is titrated to determine the optimal concentration used for 32Dkit cell growth. </td>
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measuring calpain activity in fixed and living cells by flow cytometry

钙蛋白酶是无处不在的细胞内钙敏感，中性半胱氨酸蛋白酶<sup&gt; 1</sup&gt;。钙蛋白酶发挥至关重要的作用，在许多生理过程，包括信号，细胞骨架重构，调节基因表达，细胞凋亡和细胞周期进程<sup&gt; 1</sup&gt;。钙蛋白酶有牵连的许多病症，包括肌营养不良症，癌症，糖尿病，阿尔茨海默氏症和多发性硬化症<sup&gt; 1</sup&gt;。钙蛋白酶调节是复杂的，完全了解。 mRNA和蛋白水平与活动相关的不良的限制措施蛋白酶活性基因或蛋白质表达技术的使用。此视频协议的细节在我们的测量在固定和活细胞的钙蛋白酶活性的实验室开发一个流式细胞仪测定。此方法使用中银LM - CMAC的荧光底物，这是由蛋白酶裂解，来衡量蛋白酶的活动。<sup&gt; 2</sup&gt;在这个视频中，蛋白酶活动是在固定和生活的小鼠32Dkit白血病细胞，单独或作为一个人口脾的一部分使用LSRII（BD生物科学）。 32Dkit细胞有升高的活动相比，正常脾。

Watch this Scientific Journal Video about Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry at JoVE.com

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

本文将详细测量固定和生活用流式细胞仪检测细胞中的蛋白酶活动的协议。

在固定和活细胞，流式细胞仪测量钙蛋白酶活性

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

measuring-calpain-activity-in-fixed-and-living-cells-by-flow-cytometry

Research

JoVE Journal

Immunology and Infection

11.8K 次观看.  University of Toronto. 本文将详细测量固定和生活用流式细胞仪检测细胞中的蛋白酶活动的协议。

视频: Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

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免疫与感染

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

基于细胞的流式细胞仪检测，以测量细胞毒活性

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 &#176;C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

通过流式细胞仪测量附件和流感的内在病毒在A549细胞

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

利用流细胞测定人类白细胞测定阿斯珀吉鲁斯紫锥体烟曲的噬菌体

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, transports immune complexes, and participates in humoral and cellular immune responses. CR1 is present on the surface of many cell types, including erythrocytes, and exhibits polymorphisms in length, structure (Knops, or KN, blood group), and density. The average density of CR1 per erythrocyte (CR1/E) is 500 molecules per erythrocyte. This density varies from one individual to another (100&#8211;1,200 CR1/E) and from one erythrocyte to another in the same individual. We present here a robust flow cytometry method to measure the density of CR1/E, including in subjects expressing a low density, with the help of an amplifying immunostaining system. This method has enabled us to show the lowering of CR1 erythrocyte expression in diseases such as Alzheimer&#39;s disease (AD), systemic lupus erythematosus (SLE), AIDS, or malaria.

The aim of this method is to determine the CR1 density in the erythrocytes of any subject by comparing with three subjects whose erythrocyte CR1 density is known. The method uses flow cytometry after immunostaining of the subjects&#39; erythrocytes by an anti-CR1 monoclonal antibody coupled to an amplified system using phycoerythrin (PE).

使用流细胞测定测量红细胞补充受体1

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

通过流细胞学测定，测量自然获得的噬菌体诱导恶性疟Plasmodium falciparum原虫抗体

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are widely used as a convenient cell system for analyzing SAMHD1 activity due to a low level of SAMHD1 RNA expression, leading to undetectable endogenous protein expression. Based on similar assays developed in the Stoye laboratory to characterize other retroviral restriction factors, the Bishop lab developed a two-color restriction assay to analyze SAMHD1 in U937 cells. Murine Leukaemia Virus-like particles expressing SAMHD1, alongside YFP expressed from an IRES, are used to transduce U937 cells. Cells are then treated with phorbol myristate acetate to induce differentiation to a quiescent phenotype. Following differentiation, cells are infected with HIV-1 virus-like particles expressing a fluorescent reporter. After 48 h, cells are harvested and analyzed by flow cytometry. The proportion of HIV-infected cells in the SAMHD1-expressing population is compared to that in internal control cells lacking SAMHD1. This comparison reveals a restriction ratio. SAMHD1 expression leads to a five-fold reduction in HIV infection, corresponding to a restriction ratio of 0.2. Our recent substitution of RFP for the original GFP as the reporter gene for HIV infection has facilitated flow cytometry analysis.
This assay has been successfully used to characterize the effect of amino acid substitutions on SAMHD1 restriction by transducing with viruses encoding altered SAMHD1 proteins, derived from site-directed mutagenesis of the expression vector. For example, the catalytic site substitutions HD206-7AA show a restriction phenotype of 1, indicating a loss of restriction activity. Equally, the susceptibility of different tester viruses can be determined. The assay can be further adapted to incorporate the effect of differentiation status, metabolic status, and SAMHD1 modifiers to better understand the relationship between SAMHD1, cell metabolic state, and viral restriction.

Described here is an established method to determine the extent of HIV-1 restriction by the cellular inhibitory protein SAMHD1. Human myeloid lineage U937 cells are transduced with a SAMHD1 expression vector co-expressing YFP, differentiated and then challenged with HIV-RFP. The level of restriction is determined by flow cytometry analysis.

流式细胞术分析人髓系U937细胞中SAMHD1限制性

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are ...