Apoyado por becas de los Institutos Canadienses de Investigación en Salud, la leucemia y el linfoma de la Sociedad de Canadá y la Fundación Linfoma de Canadá.

Preparar los reactivos Nota: Todas las PBS contiene Ca 2 + y Mg 2 + <ol><li> Detección de los reactivos Añadir 20μM 7-amino-4-clorometil cumarina, t-BOC-leucina-metionina amida (BOC-LM-CMAC) a temperatura ambiente PBS. Cada suspensión de células se requieren reactivos de detección de 2 ml. </li><li> Paraformaldehído al 1% (células fijas) Diluir al 4% de paraformaldehído en solución de reserva temperatura ambiente PBS. Alícuota de 1 ml de paraformaldehído al 1% a cada tubo FACS. Tres tubos de FACS se requieren para cada condición experimental (por ejemplo, 32Dkit, 32Dkit + PD150606, 32Dkit + PD98059 necesitan 9 tubos FACS) </li><li> PD150606 (inhibidor de la calpaína) Reconstituir PD150606 en DMSO a una concentración final de 100 mm. Proteja el reactivo y todas las células tratadas con ella de la luz durante la duración de este experimento. </li><li> PD98059 (inhibidor de MEK1) Reconstituir PD98059 en DMSO a una concentración final de 50 mm. </li></ol> Prepare las células <ol><li> Se cultivan las células 32Dkit a 5 x 10 5 a 1 x 10 6 células por ml de Opti-MEM suplementado con los medios de comunicación fetal al 5% de suero bovino, WEHI-3 sobrenadante como fuente de IL-3 (o cualquier otra fuente de IL-3 como como proteína recombinante), el 0,1% de 2-mercaptoetanol y antibióticos. </li><li> Tomar 12 x 10 6 células 32Dkit en tres tubos Falcon de 15 ml (4 x 10 6 células por tubo). </li><li> Que sedimenten las células a 1000 rpm durante 5 minutos a 15 ° C. </li><li> Aspirar el sobrenadante. Resuspender las células en 2 ml de PBS a temperatura ambiente. </li><li> El tratamiento de una suspensión celular con 50 micras PD150606. Asegurar que la muestra está protegido de la luz por la que cubre el tubo Falcon con papel de aluminio. Vortex brevemente y luego se incuba durante veinte a treinta minutos a temperatura ambiente en la oscuridad. </li><li> El tratamiento de la suspensión celular en segundo lugar con 20μM PD98059. Placa de las células en una placa de cultivo de tejidos de 35 mm y se incuba durante 2 horas a 37 º C. </li></ol> Calpaína ensayo en células fijas <ol><li> Mientras que las muestras se incuban con PD150606, comenzará el ensayo de la calpaína en las muestras no tratadas. Comience por la adición de reactivos de detección de 2 ml de cada suspensión celular. Inmediatamente agrega 1 ml de la suspensión celular / mezcla de detección reactivos a los tubos previamente preparado FACS con paraformaldehído al 1% (0 puntos minutos de tiempo). </li><li> Incubar las suspensiones de células con el reactivo de detección durante 5 minutos a temperatura ambiente. A continuación, añadir 1 ml de la suspensión celular a un tubo con 1 mL FACS paraformaldehído al 1%. </li><li> Continuar la incubación de las suspensiones celulares con la mezcla de detección por otros 5 minutos a temperatura ambiente. A continuación, añadir 1 ml de la suspensión celular a un tubo con 1 mL FACS paraformaldehído al 1%. </li><li> Repita el ensayo de calpaína en células 32Dkit que han sido tratados con inhibidores. </li><li> Fijar las células durante la noche en la oscuridad a 4 ° C. </li><li> Al día siguiente, que sedimenten las células a 1800 rpm durante 5 minutos a 15 ° C. </li><li> Aspirar el sobrenadante. Resuspender las células en PBS 750uL. Colocar las células en hielo y mantener en la oscuridad. </li></ol> Adquisición de datos para las células fijas <ol><li> Analizar las células utilizando un LSR II (BD Bioscience). Los filtros se establecen para el Xcyte Lightwave (UV) con láser, la excitación línea 355nm y el láser de 25mW de potencia, emisión de 405 y 450 nm para el sustrato y producto, respectivamente. Filtros de paso de banda de sustrato y de producto son 405/20 y 450/50. Filtros de paso largo por el sustrato y productos son 405 y 450. El voltaje de PMT fue creada en el 350-375 de sustrato y 325-350 para el producto. </li><li> Puesta en marcha un experimento BD FACSDiVa con los siguientes parámetros: dispersión frontal, dispersión lateral; Indo-1 Blue (log), Indo-1 Violeta (log). En la hoja de establecer los siguientes gráficos: dispersión frontal versus dispersión lateral gráfico de puntos con una puerta en las células vivas (Figura 1a), Indo-1 histograma azul, Indo-1 histograma violeta, Indo-1 Azul vs Indo-1 Violeta gráfico de puntos. </li><li> Calibrar el II LSR con AlignFlow y AlignFlow más perlas fluorescentes (Invitrogen). El voltaje de PMT debe ser ajustada para colocar el pico del histograma de fluorescencia de cuentas en el mismo canal antes de cada experimento. </li><li> El proceso de adquisición de datos utilizando las células 32Dkit en el punto 0 minutos de tiempo. Ajustar los voltajes de parámetros según sea necesario. Adquirir y registrar 10.000 eventos por muestra. </li><li> Repetir la adquisición de cada muestra, el enjuague de la máquina con tampón FACS entre cada tubo. </li><li> Exportar los datos. Asegúrese de LSR II se limpia de acuerdo a las directrices del Instituto. </li></ol> Calpaína ensayo en células vivas <ol><li> Preparar las suspensiones de células que el anterior, sin PD150606. Llevar las muestras a la II LSR y establecer filtros y parámetros que el anterior. Establecer la hoja de trabajo que el anterior. Incluya un diagrama de puntos que muestra gran Indo-1 azul en función del tiempo. Configurar el programa para adquirir el numero máximonúmero de eventos (no hay puerta de parada). </li><li> Añadir 50 micras PD150606 a una suspensión celular de las células 32Dkit. Mantener la muestra en la oscuridad y se incuban a temperatura ambiente durante 20-30 minutos. </li><li> Proceso de adquisición de datos sobre la suspensión de células 32Dkit sin PD150606. Use un caudal bajo de 200 a 300 eventos por segundo. Después de 30 segundos a 1 minuto retirar el tubo de la máquina. Añadir 20μM BOC-LM-CMAC a las células. Vortex brevemente y luego continuar la adquisición de los datos. </li><li> Adquirir datos durante 10-20 minutos o hasta que la actividad de la calpaina mesetas. </li><li> Repita los pasos 3 y 4 con las células tratadas con 32Dkit PD150606. </li><li> Exportar los datos. Asegúrese de LSR II se limpia de acuerdo a las directrices del Instituto. </li></ol> Calpaína ensayo en el complejo de las poblaciones de células (Mixto) * Este experimento identificar las células 32Dkit en una suspensión de células recolectadas a partir de un bazo de ratón. <ol><li> Cosecha del bazo de cada ratón. Lisis de los glóbulos rojos con NH 4 Cl durante 10 minutos a temperatura ambiente. </li><li> Preparar suspensiones de células individuales en 2 ml de PBS. </li><li> Dividir cada suspensión de células en dos. El tratamiento de una suspensión celular con 50 micras PD150606 durante 20-30 minutos a temperatura ambiente en la oscuridad. </li><li> Siga el procedimiento anterior para el ensayo de calpaína en células fijas (pasos 1-6) </li><li> Aspirar el sobrenadante. Lavar las células en 500μL de PBS. Aspirar el sobrenadante. </li><li> Resuspender las células en un tampón de tinción de 200μL con 2% de BSA. Incubar durante 15 minutos a temperatura ambiente. </li><li> Prepare el anticuerpo primario (FITC anti-ratón CD117). El anticuerpo se utilizó en una dilución 1:200 en buffer de tinción. Asegúrese de que el anticuerpo se mantiene en la oscuridad. </li><li> Añadir el anticuerpo primario 200μL a las células. Incubar durante 30 minutos a temperatura ambiente en la oscuridad. </li><li> Añadir 400μL de PBS para aumentar el volumen de citometría de flujo. Lugar de las células en hielo y mantener en la oscuridad. </li><li> Proceda como se describe en obtener datos para las células fijas. Añadir FITC a los parámetros e incluyen un histograma para FITC en la hoja. </li></ol> Análisis y Resultados Representante <ol><li> Analizar los datos utilizando FlowJo (Treestar). Puerta de las células en vivo basado en el perfil de dispersión frontal y lateral (Figura 1a). Determinar la media de fluorescencia para Indo-azul de la puerta de células viables (Figura 1b). </li><li> a) Para la población de células mixtas <ul><li> el uso de fluorescencia FITC en las parcelas de histogramas y un punto para identificar la población 32Dkit </li><li> Determinar la media de fluorescencia para Indo-Blue de la población 32Dkit </li></ul></li><li> Utilice Microsoft Excel para representar gráficamente la media de fluorescencia (Figura 2a). Calcular la pendiente de la línea entre el 5 y 10 minutos. Gráfico de la pendiente en forma de gráfico de barras para determinar la actividad de la calpaína en las células (Figura 2b). </li></ol><img src="/files/ftp_upload/2050/2050fig1a.jpg" alt="Figura 1a" /> Figura 1a. Determinación de células en vivo basado en el perfil de dispersión frontal y lateral. <img src="/files/ftp_upload/2050/2050fig1b.jpg" alt="Figura 1b" /> Figura 1b. El cambio promedio de fluorescencia más de 10 minutos de tiempo por supuesto. <img src="/files/ftp_upload/2050/2050fig2a.jpg" alt="Figura 2a" /> Figura 2a. Representante gráfico de la media de fluorescencia. <img src="/files/ftp_upload/2050/2050fig2b.jpg" alt="Figura 2b" /> Figura 2b. Calpaína actividad en las células con inhibidores de la 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>

La actividad de la calpaína en células 32Dkit fijos y la vida se ha determinado en este protocolo de vídeo. El tratamiento de la línea de células con el inhibidor de la calpaína PD150606 dio lugar a una inhibición completa de la actividad de la calpaína en las células. Además, el tratamiento con el inhibidor de MEK1 PD98059 dado lugar a una inhibición completa de la actividad de la calpaína en las células. ERK es un importante activador de la calpaína-2 3. Estos resultados sugieren que la calpa?...

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		<th>Material Name</th>
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<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

Calpaínas son omnipresentes intracelular, sensible calcio, cisteína proteasas neutras<sup&gt; 1</sup&gt;. Calpaínas juegan un papel crucial en muchos procesos fisiológicos, incluida la señalización, la remodelación del citoesqueleto, la regulación de la expresión génica, la apoptosis y la progresión del ciclo celular<sup&gt; 1</sup&gt;. Calpaínas han sido implicados en muchas patologías como distrofias musculares, cáncer, diabetes, enfermedad de Alzheimer y la esclerosis múltiple<sup&gt; 1</sup&gt;. Calpaína regulación es compleja y no completamente entendido. los niveles de ARNm y proteínas se correlacionan poco con la actividad, lo que limita el uso de las técnicas de la expresión de genes o proteínas para medir la actividad de la calpaína. Esta detalles del protocolo de vídeo un ensayo de citometría de flujo desarrollado en nuestro laboratorio para medir la actividad de la calpaína en células fijadas y de vida. Este método utiliza el sustrato fluorescente BOC-LM-CMAC, que se escinde específicamente por la calpaína, para medir la actividad de calpaína.<sup&gt; 2</sup&gt; En este video, la actividad de la calpaína en fijo y que viven las células murinas 32Dkit leucemia, solo o como parte de una población de esplenocitos se mide utilizando un LSRII (BD Bioscience). 32Dkit células han demostrado tener actividad elevada en comparación con esplenocitos normales.

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

Este artículo detallará el protocolo para la medición de la actividad de la calpaína en células fijadas y la vida mediante citometría de flujo.

Medición de la actividad calpaína en células fijadas y vivir por citometría de flujo

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 vistas.  University of Toronto. Este artículo detallará el protocolo para la medición de la actividad de la calpaína en células fijadas y la vida mediante citometría de flujo.

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

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Humano In Vitro Supresión como herramienta de cribado para el reconocimiento de un estado inicial de desequilibrio inmunológico

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Investigación

Inmunología e Infección

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.

A raíz de la celda de destino en<em&gt; E. coli</em&gt; Después de la infección por fagos 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 ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

El examen de selección tímica positivo y negativo por citometría de flujo

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

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.

Medición de apego y Internalización del Virus de la Influenza en células A549 por Citometría de Flujo

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.

Clasificación de células activadas por fluorescencia para la purificación de células dendríticas plasmocitoide de la médula ósea de ratones

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.

Aislamiento y cultivo In Vitro de los macrófagos alveolares murinos y humanos

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.

Preparación de la médula ósea entera para el análisis de citometría de masa de células de linaje neutrófilo

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.

Medición de la fagocitosis de Aspergillus fumigatus Conidia por leucocitos humanos utilizando citometría de flujo

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).

Medición del receptor de complemento de eritrocitos 1 mediante citometría de flujo

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