Supported by grants from the Canadian Institutes of Health Research, The Leukemia and Lymphoma Society of Canada and the Lymphoma Foundation of Canada.

Prepare Reagents
Note: All PBS contains Ca2+ and Mg2+
<ol>
 <li>Detection Reagent 
 Add 20&mu;M 7-amino-4-chloromethyl coumarin, t-BOC-Leucine-methionine amide (BOC-LM-CMAC) to room temperature PBS. Each cell suspension will require 2mL detection reagent.</li>
 <li>1% Paraformaldehyde (Fixed Cells) 
 Dilute 4% paraformaldehyde stock solution in room temperature PBS. Aliquot 1mL 1% paraformaldehyde to each FACS tube. Three FACS tubes are required for each experimental condition (ex. 32Dkit, 32Dkit + PD150606, 32Dkit + PD98059 need 9 FACS tubes)</li>
 <li>PD150606 (calpain inhibitor) 
 Reconstitute PD150606 in DMSO to a final concentration of 100mM. Protect this reagent and all cells treated with it from light for the duration of this experiment.</li>
 <li>PD98059 (MEK1 inhibitor) 
 Reconstitute PD98059 in DMSO to a final concentration of 50mM.</li>
</ol>
Prepare Cells
<ol>
 <li>Grow 32Dkit cells at 5 x 105 to 1 x 106 cells per mL in Opti-MEM media supplemented with 5% fetal bovine serum, WEHI-3 supernatant as a source of IL-3 (or any other source for IL-3 such as recombinant protein), 0.1% 2-mercaptoethanol and antibiotics.</li>
 <li>Collect 12 x 106 32Dkit cells into three 15mL Falcon tubes (4 x 106 cells per tube).</li>
 <li>Pellet the cells at 1000 RPM for 5 minutes at 15&deg;C.</li>
 <li>Aspirate the supernatant. Resuspend the cells in 2mL room temperature PBS.</li>
 <li>Treat one cell suspension with 50&mu;M PD150606. Ensure the sample is protected from light by covering the Falcon tube with aluminum foil. Vortex briefly then incubate for twenty to thirty minutes at room temperature in the dark.</li>
 <li>Treat the second cell suspension with 20&mu;M PD98059. Plate the cells in a 35mm tissue culture dish and incubate for 2 hours at 37&deg;C.</li>
</ol>
Calpain Assay in Fixed Cells
<ol>
 <li>While the samples are incubating with PD150606, begin the calpain assay on the untreated samples. Begin by adding 2mL detection reagent to each cell suspension. Immediately add 1mL of the cell suspension/detection reagent mixture to the previously prepared FACS tubes with 1% paraformaldehyde (0 minutes time point).</li>
 <li>Incubate cell suspensions with detection reagent for 5 minutes at room temperature. Then add 1mL of the cell suspension to a FACS tube with 1mL 1% paraformaldehyde.</li>
 <li>Continue incubating the cell suspensions with detection mixture for an additional 5 minutes at room temperature. Then add 1mL of the cell suspension to a FACS tube with 1mL 1% paraformaldehyde.</li>
 <li>Repeat calpain assay on 32Dkit cells that have been treated with inhibitors.</li>
 <li>Fix cells overnight in the dark at 4&deg;C.</li>
 <li>The next day pellet the cells at 1800 RPM for 5 minutes at 15&deg;C.</li>
 <li>Aspirate the supernatant. Resuspend the cells in 750uL PBS. Place cells on ice and keep in the dark.</li>
</ol>
Acquire Data for Fixed Cells
<ol>
 <li>Analyze the cells using an LSR II (BD Bioscience). Filters are set for the Lightwave Xcyte (UV) laser, excitation line 355nm and laser power 25mW, emission of 405 and 450 nm for substrate and product respectively. Band pass filters for substrate and product are 405/20 and 450/50. Long pass filters for substrate and product are 405 and 450. The PMT voltage was set on 350-375 for substrate and 325-350 for the product.</li>
 <li>Set-up a BD FACSDiva experiment with the following parameters: forward scatter; side scatter; Indo-1 Blue (log); Indo-1 Violet (log). On the worksheet set up the following graphs: forward scatter vs. side scatter dot plot with a gate on live cells (Figure 1a), Indo-1 Blue histogram, Indo-1 Violet histogram, Indo-1 Blue vs. Indo-1 Violet dot plot.</li>
 <li>Calibrate the LSR II with AlignFlow and AlignFlow plus fluorescent beads (Invitrogen). The PMT voltage should be adjusted to place the peak of the bead fluorescence histogram in the same channel prior to every experiment.</li>
 <li>Begin acquiring data using the 32Dkit cells at the 0 minute time point. Adjust parameter voltages as necessary. Acquire and record 10,000 events per sample.</li>
 <li>Repeat acquisition for each sample, rinsing the machine with FACS buffer between each tube.</li>
 <li>Export the data. Ensure LSR II is cleaned according to institute guidelines.</li>
</ol>
Calpain Assay in Live Cells
<ol>
 <li>Prepare the cell suspensions as above, with no PD150606. Bring the samples to the LSR II and set up filters and parameters as above. Set up the worksheet as above. Include a large dot plot showing Indo-1 Blue vs. time. Set the program to acquire the maximum number of events (no stop gate).</li>
 <li>Add 50&mu;M PD150606 to one cell suspension of 32Dkit cells. Keep the sample in the dark and incubate at room temperature for 20-30 minutes.</li>
 <li>Begin acquiring data on the 32Dkit cell suspension with no PD150606. Use a low flow rate of 200-300 events per second. After 30 seconds to 1 minute remove the tube from the machine. Add 20&mu;M BOC-LM-CMAC to the cells. Vortex briefly then continue acquiring the data.</li>
 <li>Acquire data for 10-20 minutes or until the calpain activity plateaus.</li>
 <li>Repeat steps 3 and 4 with 32Dkit cells treated with PD150606.</li>
 <li>Export the data. Ensure LSR II is cleaned according to institute guidelines.</li>
</ol>
Calpain Assay in Complex (Mixed) Cell Populations
*This experiment will identify 32Dkit cells in a cell suspension harvested from a mouse spleen.
<ol>
 <li>Harvest the spleen from each mouse. Lyse the red blood cell with NH4Cl for 10 minutes at room temperature.</li>
 <li>Prepare single cell suspensions in 2mL PBS.</li>
 <li>Divide each cell suspension in two. Treat 1 cell suspension with 50&mu;M PD150606 for 20-30 minutes at room temperature in the dark.</li>
 <li>Proceed as above for the Calpain Assay in Fixed Cells (Steps 1-6)</li>
 <li>Aspirate the supernatant. Wash the cells in 500&mu;L PBS. Aspirate the supernatant.</li>
 <li>Resuspend the cells in 200&mu;L staining buffer with 2% BSA. Incubate for 15 minutes at room temperature.</li>
 <li>Prepare the primary antibody (FITC anti-mouse CD117). The antibody should be used at a 1:200 dilution in staining buffer. Ensure the antibody is kept in the dark.</li>
 <li>Add 200&mu;L primary antibody to the cells. Incubate for 30 minutes at room temperature in the dark.</li>
 <li>Add 400&mu;L PBS to increase the volume for flow cytometry. Place the cells on ice and keep in the dark.</li>
 <li>Proceed as described in Acquire Data for Fixed Cells. Add FITC to the parameters and include a histogram for FITC on the worksheet.</li>
</ol>
Analysis and Representative Results
<ol>
 <li>Analyze data using FlowJo (Treestar). Gate on live cells based on forward and side scatter profile (Figure 1a). Determine the mean fluorescence for Indo-Blue from the viable cell gate (Figure 1b).</li>
 <li>a) For the Mixed Cell Population
 <ul>
 <li>use FITC fluorescence on histograms and dot plots to identify the 32Dkit population</li>
 <li>Determine the mean fluorescence for Indo-Blue of the 32Dkit population</li>
 </ul>
 </li>
 <li>Use Microsoft Excel to graph the mean fluorescence (Figure 2a). Calculate the slope of the line between 5 and 10 minutes. Graph the slope as a bar graph to determine calpain activity in the cells (Figure 2b).</li>
</ol>
<img src="/files/ftp_upload/2050/2050fig1a.jpg" alt="Figure 1a" /> 
 Figure 1a. Determining live cells based on forward and side scatter profile.
<img src="/files/ftp_upload/2050/2050fig1b.jpg" alt="Figure 1b" /> 
 Figure 1b. Mean fluorescence change over 10 minute time course.
<img src="/files/ftp_upload/2050/2050fig2a.jpg" alt="Figure 2a" /> 
 Figure 2a. Representative graph of mean fluorescence.
<img src="/files/ftp_upload/2050/2050fig2b.jpg" alt="Figure 2b" /> 
 Figure 2b. Calpain activity in 32Dkit cells with inhibitors.

<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>

No conflicts of interest declared.

The calpain activity in fixed and living 32Dkit cells has been determined in this video protocol. Treatment of the cell line with the calpain inhibitor PD150606 resulted in complete inhibition of calpain activity in the cells. In addition, treatment with the MEK1 inhibitor PD98059 resulted in complete inhibition of calpain activity in the cells. ERK is a major activator of calpain-2 3. These results suggest that calpain-2 activity is predominant in the 32Dkit cells. Current work in our laboratory is investigat...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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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>
</tr>		</tbody>
		</table>
		</div>

measuring calpain activity in fixed and living cells by flow cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

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

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

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 Görüntüleme.  University of Toronto. Bu makale, flow sitometri kullanarak sabit ve canlı hücrelerin calpain aktivitesini ölçmek için ayrıntılı protokol.

Video: 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.

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

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.

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.

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.

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.

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.

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.

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).

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).

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.

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