Soutenu par des subventions des Instituts canadiens de recherche en santé, La Société de leucémie et lymphome du Canada et la Fondation Lymphome du Canada.

Préparer des réactifs Note: Tous PBS contient Ca 2 + et Mg 2 + <ol><li> Réactif de détection Ajouter 20μM 7-amino-4-chlorométhyl coumarine, t-BOC-Leucine-méthionine amide (BOC-LM-CMAC) à la température ambiante du PBS. Chaque suspension cellulaire exigera réactif de détection 2mL. </li><li> Paraformaldéhyde 1% (cellules fixées) Diluer 4% paraformaldéhyde solution stock à la température ambiante du PBS. Paraformaldéhyde Aliquoter 1mL de 1% à chaque tube FACS. Trois tubes FACS sont requis pour chaque condition expérimentale (ex. 32Dkit, 32Dkit + PD150606, 32Dkit + PD98059 besoin de 9 tubes FACS) </li><li> PD150606 (inhibiteur de calpaïne) Reconstituer PD150606 dans le DMSO à une concentration finale de 100 mM. Protégez ce réactif et de toutes les cellules traitées avec elle de la lumière pendant la durée de cette expérience. </li><li> PD98059 (MEK1 inhibiteur) Reconstituer PD98059 dans le DMSO à une concentration finale de 50 mM. </li></ol> Préparer les cellules <ol><li> Cultiver des cellules 32Dkit à 5 x 10 5 à 1 x 10 6 cellules par millilitre dans les médias Opti-MEM supplémenté avec 5% de sérum bovin fœtal, WEHI-3 surnageant comme une source d&#39;IL-3 (ou toute autre source d&#39;IL-3 tels comme protéine recombinante), 0,1% de 2-mercaptoéthanol et des antibiotiques. </li><li> Prélever 12 x 10 6 cellules dans des tubes Falcon 32Dkit trois 15mL (4 x 10 6 cellules par tube). </li><li> Pellet les cellules à 1000 rpm pendant 5 minutes à 15 ° C. </li><li> Aspirer le surnageant. Resuspendre les cellules dans 2 ml de PBS température ambiante. </li><li> Traiter une suspension cellulaire avec 50 microns PD150606. S&#39;assurer que l&#39;échantillon est protégé de la lumière en couvrant le tube Falcon de papier d&#39;aluminium. Vortex brièvement puis incuber pendant vingt à trente minutes à température ambiante dans l&#39;obscurité. </li><li> Traiter la suspension cellulaire seconde avec 20μM PD98059. Assiette des cellules dans une boîte de culture tissulaire de 35 mm et incuber pendant 2 heures à 37 ° C. </li></ol> Assay calpaïne dans des cellules fixées <ol><li> Alors que les échantillons sont incubation avec PD150606, commencer le dosage calpaïne sur les échantillons non traités. Commencez par ajouter 2mL réactif de détection à chaque suspension cellulaire. Immédiatement ajouter 1ml de la suspension cellulaire / mélange de réactifs de détection pour les tubes préalablement préparé FACS avec du paraformaldéhyde 1% (0 point dans le temps minutes). </li><li> Incuber les suspensions cellulaires avec le réactif de détection pendant 5 minutes à température ambiante. Puis ajoutez 1 ml de la suspension cellulaire dans un tube à FACS avec 1mL paraformaldéhyde 1%. </li><li> Poursuivre l&#39;incubation des suspensions cellulaires avec le mélange de détection pour un supplément de 5 minutes à température ambiante. Puis ajoutez 1 ml de la suspension cellulaire dans un tube à FACS avec 1mL paraformaldéhyde 1%. </li><li> Répéter test calpaïne sur les cellules 32Dkit qui ont été traités avec des inhibiteurs. </li><li> Fixer les cellules pendant la nuit dans l&#39;obscurité à 4 ° C. </li><li> Le lendemain un culot cellulaire à 1800 rpm pendant 5 minutes à 15 ° C. </li><li> Aspirer le surnageant. Resuspendre les cellules dans 750uL PBS. Placez les cellules sur la glace et de garder dans l&#39;obscurité. </li></ol> Acquérir des données pour des cellules fixées <ol><li> Analyser les cellules en utilisant une II LSR (BD Bioscience). Les filtres sont fixés pour l&#39;Xcyte Lightwave (UV) au laser, ligne d&#39;excitation 355nm 25mW et laser de puissance, l&#39;émission de 405 et 450 nm pour le substrat et le produit respectivement. Filtres passe-bande pour le substrat et de produit sont 405/20 450/50 et. Filtres passe longue pour le substrat et de produit sont 405 et 450. La tension a été mis sur le PMT 350-375 pour le substrat et 325-350 pour le produit. </li><li> Mise en place d&#39;une expérience BD FACSDiva avec les paramètres suivants: diffusion vers l&#39;avant; diffusion latérale; indo-1 Blue (log); Indo-1 Violet (log). Sur la feuille de calcul mis en place les graphiques suivants: diffusion vers l&#39;avant vs dot plot côté de dispersion avec une porte sur des cellules vivantes (figure 1a), indo-1 histogramme bleu, indo-1 histogramme Violet, indo-1 Blue vs indo-1 Violet dot plot. </li><li> Calibrer le LSR II avec AlignFlow et AlignFlow ainsi perles fluorescentes (Invitrogen). La tension PMT devrait être ajusté à placer le pic de l&#39;histogramme de fluorescence billes dans le même canal avant chaque expérience. </li><li> Commencer à acquérir des données en utilisant les cellules 32Dkit au point 0 minutes de temps. Ajustez les tensions paramètre comme nécessaire. Acquérir et enregistrer 10.000 manifestations par échantillon. </li><li> Répétez l&#39;acquisition pour chaque échantillon, le rinçage de la machine avec un tampon de FACS entre chaque tube. </li><li> Exporter les données. Assurez-LSR II est nettoyé conformément aux directives de l&#39;institut. </li></ol> Assay calpaïne dans des cellules vivantes <ol><li> Préparer les suspensions de cellules comme ci-dessus, sans PD150606. Apportez les échantillons au LSR II et mettre en place des filtres et des paramètres comme ci-dessus. Mettre en place la feuille de calcul que ci-dessus. Inclure une dot plot grand format indo-1 bleu en fonction du temps. Régler le programme d&#39;acquisition de la NUM maximalenombre d&#39;événements (pas de porte d&#39;arrêt). </li><li> Ajouter 50 microns PD150606 à une suspension cellulaire de cellules 32Dkit. Conserver l&#39;échantillon dans l&#39;obscurité et incuber à température ambiante pendant 20-30 minutes. </li><li> Commencer à acquérir des données sur la suspension cellulaire 32Dkit sans PD150606. Utilisez un faible débit de 200-300 événements par seconde. Après 30 secondes à 1 minute, retirer le tube de la machine. Ajouter 20μM BOC-LM-CMAC dans les cellules. Vortex brièvement puis continuer l&#39;acquisition des données. </li><li> Acquérir des données pendant 10-20 minutes ou jusqu&#39;à ce que les plateaux d&#39;activité calpaïne. </li><li> Répétez les étapes 3 et 4 avec des cellules traitées avec 32Dkit PD150606. </li><li> Exporter les données. Assurez-LSR II est nettoyé conformément aux directives de l&#39;institut. </li></ol> Assay calpaïne dans le Complexe populations cellulaires (Mixte) * Cette expérience permettra d&#39;identifier les cellules 32Dkit dans une suspension de cellules récoltées à partir d&#39;un rate de souris. <ol><li> Récolte de la rate de chaque souris. Lyse des globules rouges avec NH 4 Cl pendant 10 minutes à température ambiante. </li><li> Préparer les suspensions de cellules simples dans 2mL de PBS. </li><li> Diviser chaque suspension cellulaire en deux. Traiter une suspension cellulaire avec 50 microns PD150606 pendant 20-30 minutes à température ambiante dans l&#39;obscurité. </li><li> Procédez comme précédemment pour le dosage de la calpaïne dans des cellules fixes (étapes 1-6) </li><li> Aspirer le surnageant. Laver les cellules dans 500μL de PBS. Aspirer le surnageant. </li><li> Resuspendre les cellules dans un tampon de coloration 200 pl avec BSA 2%. Incuber pendant 15 minutes à température ambiante. </li><li> Préparer l&#39;anticorps primaire (FITC anti-souris CD117). L&#39;anticorps doit être utilisé à une dilution 1:200 dans la coloration de mémoire tampon. Assurer l&#39;anticorps est maintenu dans l&#39;obscurité. </li><li> Ajouter 200 ul d&#39;anticorps primaires pour les cellules. Incuber pendant 30 minutes à température ambiante dans l&#39;obscurité. </li><li> Ajouter 400μL de PBS pour augmenter le volume pour la cytométrie de flux. Placez les cellules sur la glace et de garder dans l&#39;obscurité. </li><li> Procédez comme décrit dans l&#39;acquisition de données pour les cellules fixes. Ajouter FITC aux paramètres et notamment un histogramme pour FITC sur la feuille de calcul. </li></ol> Analyse et résultats représentatifs <ol><li> Analyser les données en utilisant FlowJo (Treestar). Porte sur des cellules vivantes en fonction du profil de dispersion vers l&#39;avant et de côté (figure 1a). Déterminer la moyenne de fluorescence pour les Indo-bleu de la porte de cellules viables (figure 1b). </li><li> a) Pour la population de cellules mixtes <ul><li> l&#39;utilisation fluorescence FITC sur des parcelles histogrammes et dot pour identifier la population 32Dkit </li><li> Déterminer la moyenne de fluorescence pour les Indo-bleu de la population 32Dkit </li></ul></li><li> Utilisez Microsoft Excel pour représenter graphiquement la moyenne de fluorescence (figure 2a). Calculer la pente de la ligne entre 5 et 10 minutes. Graphique de la pente comme un diagramme pour déterminer l&#39;activité calpaïne dans les cellules (Figure 2b). </li></ol><img src="/files/ftp_upload/2050/2050fig1a.jpg" alt="Figure 1a" /> Figure 1a. Déterminer les cellules vivantes en fonction du profil de dispersion avant et latérale. <img src="/files/ftp_upload/2050/2050fig1b.jpg" alt="Figure 1b" /> Figure 1b. Variation moyenne de fluorescence plus de 10 cours du temps minute. <img src="/files/ftp_upload/2050/2050fig2a.jpg" alt="Figure 2a" /> Figure 2a. Représentant graphique de la fluorescence moyenne. <img src="/files/ftp_upload/2050/2050fig2b.jpg" alt="Figure 2b" /> Figure 2b. Activité de la calpaïne dans les cellules avec des inhibiteurs 32Dkit.

<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>

L&#39;activité calpaïne dans les cellules 32Dkit fixe et la vie a été déterminée dans ce protocole vidéo. Traitement de la lignée cellulaire avec l&#39;inhibiteur de calpaïnes PD150606 entraîné une inhibition complète de l&#39;activité calpaïne dans les cellules. En outre, le traitement avec l&#39;inhibiteur de MEK1 PD98059 entraîné une inhibition complète de l&#39;activité calpaïne dans les cellules. ERK est un important activateur de la calpaïne-2 3. Ces résultats suggèrent que l&#39;a...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</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

Les calpaïnes sont omniprésents intracellulaire, sensible au calcium, cystéine-protéases neutres<sup&gt; 1</sup&gt;. Les calpaïnes jouent un rôle crucial dans de nombreux processus physiologiques, y compris la signalisation, le remodelage du cytosquelette, la régulation de l&#39;expression des gènes, l&#39;apoptose et la progression du cycle cellulaire<sup&gt; 1</sup&gt;. Les calpaïnes ont été impliqués dans de nombreuses pathologies, y compris les dystrophies musculaires, cancer, diabète, maladie d&#39;Alzheimer et la sclérose en plaques<sup&gt; 1</sup&gt;. Calpaïne la réglementation est complexe et incomplètement compris. les niveaux d&#39;ARNm et de protéine faible corrélation avec l&#39;activité, limitant l&#39;utilisation de techniques d&#39;expression de gènes ou de protéines pour mesurer l&#39;activité des calpaïnes. Ce protocole décrit une vidéo cytométrie en flux développé dans notre laboratoire pour mesurer l&#39;activité calpaïne dans les cellules fixes et vivant. Cette méthode utilise le substrat fluorescent BOC-LM-CMAC, qui est clivée spécifiquement par la calpaïne, pour mesurer l&#39;activité des calpaïnes.<sup&gt; 2</sup&gt; Dans cette vidéo, l&#39;activité calpaïne dans fixes et vivant des cellules de leucémie murine 32Dkit, seul ou comme partie d&#39;une population de splénocytes est mesuré en utilisant une LSRII (BD Bioscience). Cellules sont 32Dkit montré une activité élevée par rapport aux splénocytes normaux.

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

Cet article détaillera le protocole de mesure de l&#39;activité calpaïne dans les cellules fixes et de vie en utilisant la cytométrie de flux.

Mesure de l&#39;activité calpaïne dans des cellules fixées et de la Vie par cytométrie en flux

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

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Immunology and Infection

11.7K vues.  University of Toronto. Cet article détaillera le protocole de mesure de l'activité calpaïne dans les cellules fixes et de vie en utilisant la cytométrie de flux.

Vidéo: 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.

Après Cell-sort<em&gt; E. coli</em&gt; Après infection par le 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 ...

Recherche

Immunologie et infection

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.

Mesurer l&#39;attachement et l&#39;internalisation du virus grippal A dans les cellules A549 par cytométrie en flux

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-tri cellulaire activé pour la purification de cellules dendritiques plasmacytoïdes de la moelle osseuse de souris

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.

Isolement et la Culture In Vitro de Macrophages alvéolaires murines et humaines

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.

Préparation de la moelle osseuse entière pour l'analyse de la cytométrie de masse des cellules de lignée neutrophile

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.

Mesure de la phagocytose d'Aspergillus fumigatus Conidia par des leucocytes humains à l'aide de la cytométrie du flux

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).

Mesurer le récepteur de complément d’érythrocyte 1 à l’aide de la cytométrie des débits

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).

Mesure des anticorps naturels acquis de phagocytose induisant des parasites de plasmodium falciparum par un essai basé sur la cytométrie de flux

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.

Analyse de la restriction SAMHD1 par cytométrie en flux dans les cellules myéloïdes humaines U937

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