Suportada por concessões do Canadian Institutes of Health Research, The Leukemia and Lymphoma Society do Canadá e da Fundação Linfoma do Canadá.

Prepare Reagentes Nota: Todos os PBS contém Ca 2 + e Mg 2 + <ol><li> Reagente de detecção Adicionar 20Hm 7-amino-4-clorometil cumarina, t-BOC-Leucina-metionina amida (BOC-LM-CMAC) a temperatura ambiente PBS. Cada suspensão de células exigirá reagente de detecção 2 ml. </li><li> Paraformaldeído 1% (células fixas) Diluir solução stock 4% paraformaldeído em PBS à temperatura ambiente. Paraformaldeído 1mL alíquota de 1% a cada tubo FACS. Três tubos FACS são necessários para cada condição experimental (ex. 32Dkit, 32Dkit + PD150606, 32Dkit + PD98059 precisará de 9 tubos FACS) </li><li> PD150606 (inibidor da calpaína) Reconstituir PD150606 em DMSO para uma concentração final de 100mM. Proteger este reagente e todas as células tratadas com da luz para a duração do experimento. </li><li> PD98059 (MEK1 inibidor) Reconstituir PD98059 em DMSO para uma concentração final de 50mM. </li></ol> Prepare Células <ol><li> Cultivar células 32Dkit menos 5 x 10 5 a 1 x 10 6 células por ml em Opti-MEM meio suplementado com 5% de soro fetal bovino, WEHI-3 sobrenadante como fonte de IL-3 (ou qualquer outra fonte de IL-3, tais como a proteína recombinante), 0,1% 2-mercaptoetanol e antibióticos. </li><li> Coletar 12 x 10 6 células 32Dkit em três tubos de 15mL Falcon (4 x 10 6 células por tubo). </li><li> Agregar as células a 1000 RPM por 5 minutos a 15 ° C. </li><li> Aspirar o sobrenadante. Ressuspender as células em 2 mL PBS à temperatura ambiente. </li><li> Tratar uma suspensão de células com 50 ìm PD150606. Certifique-se a amostra é protegido da luz, cobrindo o tubo Falcon com papel alumínio. Vortex brevemente, em seguida, incubar por 20-30 minutos em temperatura ambiente no escuro. </li><li> Tratar da suspensão de células em segundo lugar com 20Hm PD98059. Placa de células em um prato de 35 milímetros de cultura de tecidos e incubar por 2 horas a 37 ° C. </li></ol> Calpaína Ensaio em células fixas <ol><li> Enquanto as amostras são incubadas com PD150606, começa o ensaio calpaína nas amostras não tratadas. Comece por adicionar o reagente de detecção de 2 ml para cada suspensão de células. Imediatamente adicionar 1mL da suspensão de células / mistura de reagentes de detecção para os tubos previamente preparado FACS com paraformaldeído 1% (0 ponto tempo minutos). </li><li> Incubar suspensões de células com o reagente de detecção por 5 minutos em temperatura ambiente. Em seguida, adicione 1 mL da suspensão de células para um tubo com 1 mL FACS paraformaldeído 1%. </li><li> Continue a incubar a suspensões de células com a mistura de detecção para um adicional de 5 minutos à temperatura ambiente. Em seguida, adicione 1 mL da suspensão de células para um tubo com 1 mL FACS paraformaldeído 1%. </li><li> Repita ensaio calpaína em células 32Dkit que foram tratados com inibidores. </li><li> Corrigir as células durante a noite no escuro a 4 ° C. </li><li> No dia seguinte, agregar as células a 1800 RPM por 5 minutos a 15 ° C. </li><li> Aspirar o sobrenadante. Ressuspender as células em 750uL PBS. Coloque as células no gelo e manter no escuro. </li></ol> Aquisição de dados para células fixas <ol><li> Analisar as células usando um LSR II (BD Bioscience). Os filtros são definidos para o Xcyte Lightwave (UV) laser, linha de excitação 355nm e potência do laser 25mW, emissão de 405 e 450 nm para substrato e produto, respectivamente. Filtros de banda passante para substrato e produto são 405/20 e 450/50. Filtros de passe longo para substrato e produto são 405 e 450. A tensão PMT foi fixado em 350-375 para o substrato e 325-350 para o produto. </li><li> Set-up um experimento FACSDiva BD com os seguintes parâmetros: dispersão para a frente; dispersão lateral; Indo-1 Blue (log); Indo-1 Violet (log). Na planilha configurar os seguintes gráficos: dispersão frente vs lado gráfico de pontos de dispersão com um portão de células vivas (Figura 1a), Indo-1 histograma Azul, Indo-1 histograma Violet, Indo-1 Blue vs Violet Indo-1 gráfico de pontos. </li><li> Calibrar o II LSR com AlignFlow e AlignFlow mais partículas fluorescentes (Invitrogen). A tensão PMT deve ser ajustada para colocar o pico do histograma talão de fluorescência no mesmo canal antes de cada experimento. </li><li> Começar a adquirir os dados usando as células 32Dkit no ponto de tempo 0 minuto. Ajustar tensões parâmetro como necessário. Adquirir e registrar 10 mil eventos por amostra. </li><li> Repita aquisição para cada amostra, enxaguar a máquina com tampão FACS entre cada tubo. </li><li> Exportar os dados. Garantir LSR II é limpo de acordo com as diretrizes do instituto. </li></ol> Calpaína Ensaio em células vivas <ol><li> Prepare as suspensões de células como acima, sem PD150606. Trazer as amostras para o II LSR e configurar filtros e parâmetros acima. Configure a planilha como acima. Incluir um gráfico de pontos grande mostrando Indo-1 Blue vs tempo. Definir o programa para adquirir o máximo de númerosmero de eventos (sem portão stop). </li><li> Adicionar 50 ìm suspensão de células PD150606 a uma das células 32Dkit. Manter a amostra no escuro e incubar a temperatura ambiente por 20-30 minutos. </li><li> Começar a adquirir dados sobre a suspensão de células 32Dkit sem PD150606. Use uma baixa taxa de fluxo de 200-300 eventos por segundo. Após 30 segundos a 1 minuto retire o tubo da máquina. Adicionar 20Hm BOC-LM-CMAC para as células. Vortex brevemente em seguida, continuar a aquisição dos dados. </li><li> Aquisição de dados por 10-20 minutos ou até os planaltos atividade calpaína. </li><li> Repita os passos 3 e 4 com as células tratadas com 32Dkit PD150606. </li><li> Exportar os dados. Garantir LSR II é limpo de acordo com as diretrizes do instituto. </li></ol> Calpaína Assay no Complexo Populações (Mixed) Célula * Esta experiência vai identificar as células 32Dkit em uma suspensão de células colhidas de um baço mouse. <ol><li> Colheita do baço de cada rato. Lisar as células vermelhas do sangue com NH 4 Cl por 10 minutos em temperatura ambiente. </li><li> Preparar suspensões única célula em 2 mL de PBS. </li><li> Divida cada suspensão de células em dois. Tratar uma suspensão de células com 50 ìm PD150606 por 20-30 minutos em temperatura ambiente no escuro. </li><li> Proceda como acima para o ensaio calpaína em células Fixo (Passos 1-6) </li><li> Aspirar o sobrenadante. Lavar as células em 500μL de PBS. Aspirar o sobrenadante. </li><li> Ressuspender as células em tampão coloração 200μL com BSA 2%. Incubar por 15 minutos em temperatura ambiente. </li><li> Prepare o anticorpo primário (anti-camundongo FITC CD117). O anticorpo deve ser utilizada na diluição de 1:200 na coloração buffer. Certifique-se o anticorpo é mantido no escuro. </li><li> Adicionar 200μL anticorpo primário para as células. Incubar por 30 minutos em temperatura ambiente no escuro. </li><li> Adicionar 400μL de PBS para aumentar o volume de citometria de fluxo. Coloque as células no gelo e manter no escuro. </li><li> Proceda conforme descrito em adquirir dados para Células Fixo. Adicionar FITC aos parâmetros e incluem um histograma para FITC na planilha. </li></ol> Análise e Resultados Representante <ol><li> Analisar os dados usando programa FlowJo (Treestar). Portão em células vivas com base no perfil de dispersão para a frente e lateral (Figura 1a). Determinar a fluorescência significa para Indo-Azul a partir do portão de células viáveis ​​(Figura 1b). </li><li> a) Para a população mista de células <ul><li> uso de fluorescência FITC em parcelas de histogramas e de ponto para identificar a população 32Dkit </li><li> Determinar a fluorescência significa para Indo-Azul da população 32Dkit </li></ul></li><li> Use Microsoft Excel para o gráfico de fluorescência média (Figura 2a). Calcule a inclinação da linha entre 5 e 10 minutos. Inclinação do gráfico como um gráfico de barras para determinar a atividade calpaína nas células (Figura 2b). </li></ol><img src="/files/ftp_upload/2050/2050fig1a.jpg" alt="Figura 1a" /> Figura 1a. Determinação de células vivas com base no perfil de dispersão para a frente e lateral. <img src="/files/ftp_upload/2050/2050fig1b.jpg" alt="Figura 1b" /> Figura 1b. Variação média de fluorescência mais de 10 minutos curso de tempo. <img src="/files/ftp_upload/2050/2050fig2a.jpg" alt="Figura 2a" /> Figura 2a gráfico. Representante da fluorescência média. <img src="/files/ftp_upload/2050/2050fig2b.jpg" alt="Figura 2b" /> Figura 2b. Calpaína atividade em células 32Dkit com inibidores.

<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>

Não há conflitos de interesse declarados.

A atividade de calpaína em células 32Dkit fixa e vivendo foi determinado neste protocolo de vídeo. Tratamento da linhagem de células com o inibidor de calpaína PD150606 resultou em inibição total da atividade calpaína nas células. Além disso, o tratamento com o inibidor MEK1 PD98059 resultou em inibição total da atividade calpaína nas células. ERK é um ativador principal de calpaína-2 3. Estes resultados sugerem que calpaína-2 é a atividade predominante nas células 32Dkit. Trabalhos em curso em nosso laboratório está investigando as conseqüências da atividade calpaína aumentou e as contribuições de calpaína-1 e calpaína-2 em leucemia. Este protocolo de detalhes o primeiro ensaio de citometria de fluxo com base para medir os níveis de atividade das calpaínas em células fixas e vida e pode ser usado para entender o mecanismo de regulação calpaína e seu papel nos processos patológicos utilizando células intactas. Este protocolo pode ser modificado para examinar os efeitos de inibidores ou manipulações gene calpaína sobre a atividade. Além disso, este ensaio pode ser utilizado para medir a atividade das calpaínas em uma mistura complexa de células, tais como sangue do cordão umbilical ou para identificar e medir a atividade das calpaínas em subpopulações de células isoladas do baço. Este ensaio não faz distinção entre a atividade gerada por diferentes isoformas da calpaína. Reagentes genéticas ou farmacológicas que derrubar ou inibir calpains específico pode ser usado para abordar esta questão.

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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>
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<td>AlignFlow, AlignFlow plus fluorescent beads FACS tubes</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<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 são onipresentes intracelular, cálcio-sensíveis, cisteína proteases neutras<sup&gt; 1</sup&gt;. Calpaínas desempenham um papel crucial em muitos processos fisiológicos, incluindo a sinalização, remodelação do citoesqueleto, a regulação da expressão gênica da apoptose e progressão do ciclo celular<sup&gt; 1</sup&gt;. Calpaínas tem sido implicado em muitas patologias, incluindo distrofias musculares, câncer, diabetes, doença de Alzheimer e esclerose múltipla<sup&gt; 1</sup&gt;. Calpaína regulamentação é complexo e incompletamente compreendida. níveis de mRNA e proteína correlacionam mal com a atividade, limitando o uso de técnicas de expressão do gene ou proteína para medir a atividade calpaína. Esta detalhes protocolo de vídeo de um ensaio de citometria de fluxo desenvolvido em nosso laboratório para medir a atividade das calpaínas em células fixas e de viver. Este método usa o substrato fluorescente BOC-LM-CMAC, que é clivada especificamente pela calpaína, para medir a atividade calpaína.<sup&gt; 2</sup&gt; Neste vídeo, calpaína atividade em fixos e células vivas 32Dkit murine leukemia, sozinho ou como parte de uma população esplenócitos é medida usando um LSRII (BD Bioscience). Células 32Dkit mostraram que têm atividade elevada em comparação com esplenócitos normal.

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 artigo irá detalhar o protocolo para medir a atividade das calpaínas em células fixas e vivendo por citometria de fluxo.

Medir a atividade das calpaínas em células fixas e Vivendo por citometria de fluxo

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

11.8K visualizações.  University of Toronto. Este artigo irá detalhar o protocolo para medir a atividade das calpaínas em células fixas e vivendo por citometria de fluxo.

Vídeo: 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 Supressão como ferramenta de triagem para o reconhecimento de um Estado precoce de desequilíbrio imunológico

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

Imunologia e Infecção

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.

Seguindo o destino celular, em<em&gt; E. coli</em&gt; Após a infecção por um fago 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.

Exame de seleção positiva e negativa Tímico por citometria de fluxo

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.

Baseada em células de Citometria de Fluxo Ensaio medir citotóxicos Atividade

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.

Medindo Anexo e Internalização do Vírus da Influenza A em células A549 por citometria de fluxo

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.

Classificação de células activadas por fluorescência Para a purificação de células dendríticas plasmocitï¿½des do rato de Medula Óssea

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.

Isolamento e cultura In Vitro de macrófagos alveolares murino e 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.

Preparação de medula óssea inteira para análise de citometria de massa de células de linhagem de neutrófilos

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.

Medição de phagocitose de Aspergillus fumigatus Conidia por Leukocytes Humanos usando citometria flow

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).

Medição do receptor complemento eririotcyte 1 usando citometria de fluxo

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