Supportato da sovvenzioni da parte del Canadian Institutes of Health Research, The Leukemia and Lymphoma Society of Canada e la Fondazione Linfoma del Canada.

Preparare Reagenti Nota: Tutti i PBS contiene Ca 2 + e Mg 2 + <ol><li> Rilevazione dei reagenti Aggiungi 20μM 7-ammino-4-clorometil cumarina, t-BOC-Leucina-metionina ammide (BOC-LM-CMAC) PBS a temperatura ambiente. Ogni sospensione cellulare richiederà reagente 2 ml di rilevamento. </li><li> 1% paraformaldeide (Cellule fisso) Diluire soluzione al 4% paraformaldeide magazzino a temperatura ambiente PBS. Aliquota paraformaldeide 1 ml 1% a ciascuna provetta FACS. Tre tubi FACS sono necessari per ogni condizione sperimentale (es. 32Dkit, 32Dkit + PD150606, 32Dkit + PD98059 necessari 9 tubi FACS) </li><li> PD150606 (inibitore della calpaina) Ricostituire PD150606 in DMSO ad una concentrazione finale di 100 mm. Proteggere questo reagente e tutte le cellule trattate con il medicinale dalla luce per tutta la durata di questo esperimento. </li><li> PD98059 (MEK1 inibitore) Ricostituire PD98059 in DMSO ad una concentrazione finale di 50mM. </li></ol> Preparare Cellule <ol><li> Crescere le cellule 32Dkit a 5 x 10 5 a 1 x 10 6 cellule per ml di Opti-MEM dei media integrato con 5% di siero fetale bovino, WEHI-3 surnatante come fonte di IL-3 (o qualsiasi altra fonte di IL-3 come come proteina ricombinante), 0,1% 2-mercaptoetanolo e antibiotici. </li><li> Colleziona 12 x 10 6 cellule 32Dkit in tre tubi Falcon 15 ml (4 x 10 6 cellule per tubo). </li><li> Agglomerare le cellule a 1000 giri per 5 minuti a 15 ° C. </li><li> Aspirare il sopranatante. Risospendere le cellule in 2 ml di PBS a temperatura ambiente. </li><li> Trattare un sospensione cellulare con 50 micron PD150606. Assicurarsi che il campione è al riparo dalla luce, coprendo il tubo Falcon con un foglio di alluminio. Vortex brevemente poi incubare per 20-30 minuti a temperatura ambiente al buio. </li><li> Trattare la seconda sospensione cella con 20μM PD98059. Piastra le cellule in una capsula di 35 millimetri coltura e incubare per 2 ore a 37 ° C. </li></ol> Saggio calpaina in cellule fissate <ol><li> Mentre i campioni sono incubazione con PD150606, iniziare il test calpaina sui campioni non trattati. Iniziare con l&#39;aggiunta di reagente 2 ml di rilevamento per ogni sospensione cellulare. Immediatamente aggiungere 1 ml di sospensione cellulare / miscela dei reagenti di rilevamento per i tubi precedentemente preparato FACS con 1% paraformaldeide (0 minuti di tempo punto). </li><li> Incubare sospensioni cellulari con il reagente di rilevamento per 5 minuti a temperatura ambiente. Quindi aggiungere 1 ml di sospensione cellulare ad un tubo FACS con 1 ml di paraformaldeide 1%. </li><li> Continua incubando le sospensioni delle cellule con la miscela di rilevamento per altri 5 minuti a temperatura ambiente. Quindi aggiungere 1 ml di sospensione cellulare ad un tubo FACS con 1 ml di paraformaldeide 1%. </li><li> Ripetere il test calpaina sulle cellule 32Dkit che sono stati trattati con inibitori. </li><li> Fissare le cellule durante la notte al buio a 4 ° C. </li><li> Il giorno dopo agglomerare le cellule a 1800 giri per 5 minuti a 15 ° C. </li><li> Aspirare il sopranatante. Risospendere le cellule in 750uL PBS. Luogo cellule su ghiaccio e tenere al buio. </li></ol> Acquisire dati per celle fisse <ol><li> Analizzare le cellule utilizzando un II LSR (BD Bioscience). I filtri sono impostati per la Xcyte Lightwave (UV) laser, eccitazione 355nm linea e laser di potenza 25mW, emissione di 405 e 450 nm per il substrato e di prodotto, rispettivamente. Filtri passa banda per il substrato e di prodotto sono 405/20 e 450/50. Filtri passa lungo per il substrato e di prodotto sono 405 e 450. La tensione PMT è stato impostato sul 350-375 e 325-350 per il supporto per il prodotto. </li><li> Set-up un esperimento BD FACSDiva con i seguenti parametri: scatter in avanti; side scatter, Indo-1 Blu (log), Indo-1 Viola (log). Nel foglio di lavoro istituiti i seguenti grafici: forward scatter scatter plot vs lato dot con un cancello su cellule vive (Figura 1a), Indo-1 istogramma blu, Indo-1 istogramma Viola, Indo-1 vs Blu Indo-1 Viola dot plot. </li><li> Calibrare la II LSR con AlignFlow e AlignFlow più perline fluorescenti (Invitrogen). La tensione PMT deve essere regolato per posizionare il picco dell&#39;istogramma tallone fluorescenza nel canale stesso prima di ogni esperimento. </li><li> Iniziare l&#39;acquisizione dati utilizzando le cellule 32Dkit al punto 0 minuti di tempo. Regolare tensioni parametro, se necessario. Acquisire e registrare 10.000 eventi per ogni campione. </li><li> Ripetere l&#39;acquisizione per ogni campione, il risciacquo la macchina con il tampone di FACS tra ciascun tubo. </li><li> Esportare i dati. Garantire LSR II viene pulito secondo le linee guida dell&#39;istituto. </li></ol> Saggio calpaina in cellule vive <ol><li> Preparare le sospensioni cellulari come sopra, senza PD150606. Portare i campioni al II LSR e impostare filtri e parametri come sopra. Impostare il foglio di lavoro come sopra. Includono un grande appezzamento dot mostrando Indo-1 blu in funzione del tempo. Impostare il programma di acquisire il numero massimomero di eventi (non cancello stop). </li><li> Aggiungi 50 micron PD150606 ad una sospensione cellulare di cellule 32Dkit. Tenere il campione al buio e incubare a temperatura ambiente per 20-30 minuti. </li><li> Iniziare l&#39;acquisizione dati sulla sospensione cellulare 32Dkit senza PD150606. Utilizzare un basso tasso di flusso di 200-300 eventi al secondo. Dopo 30 secondi a 1 minuto togliere il tubo dalla macchina. Aggiungi 20μM BOC-LM-CMAC alle cellule. Vortex brevemente poi proseguire l&#39;acquisizione dei dati. </li><li> Acquisire dati per 10-20 minuti o fino a quando gli altipiani attività calpaina. </li><li> Ripetere i passaggi 3 e 4 con le cellule trattate con 32Dkit PD150606. </li><li> Esportare i dati. Garantire LSR II viene pulito secondo le linee guida dell&#39;istituto. </li></ol> Saggio calpaina in complesso (Misto) popolazioni di cellule * Questo esperimento 32Dkit identificare le cellule in una sospensione di cellule prelevate da una milza del mouse. <ol><li> Raccolto la milza da ogni mouse. Lisare il globulo rosso con NH 4 Cl per 10 minuti a temperatura ambiente. </li><li> Preparare sospensioni singola cella in 2 ml di PBS. </li><li> Dividere ogni sospensione di cellule in due. Trattare 1 sospensione cellulare con 50 micron PD150606 per 20-30 minuti a temperatura ambiente al buio. </li><li> Procedere come sopra per il saggio Calpain nelle celle fisso (Fasi 1-6) </li><li> Aspirare il sopranatante. Lavare le cellule in 500μL PBS. Aspirare il sopranatante. </li><li> Risospendere le cellule in tampone di colorazione 200μL con il 2% BSA. Incubare per 15 minuti a temperatura ambiente. </li><li> Preparare l&#39;anticorpo primario (FITC anti-topo CD117). L&#39;anticorpo deve essere utilizzato a una diluizione 1:200 in colorazione buffer. Assicurarsi che l&#39;anticorpo è tenuto all&#39;oscuro. </li><li> Aggiungi 200μL anticorpo primario per le cellule. Incubare per 30 minuti a temperatura ambiente al buio. </li><li> Aggiungi 400μL PBS per aumentare il volume di citometria a flusso. Posizionare le cellule in ghiaccio e tenere al buio. </li><li> Procedere come descritto nella acquisizione dei dati per le celle fisso. Aggiungi FITC ai parametri e comprendono un istogramma per FITC sul foglio di lavoro. </li></ol> Analisi e risultati rappresentativi <ol><li> Analizzare i dati utilizzando FlowJo (Treestar). Cancello su cellule vive in base al profilo scatter in avanti e laterale (Figura 1a). Determinare la fluorescenza media per indo-blu dalla porta delle cellule vitali (Figura 1b). </li><li> a) Per la popolazione di cellule miste <ul><li> uso FITC fluorescenza su parcelle istogrammi e punto per identificare la popolazione 32Dkit </li><li> Determinare la fluorescenza media per indo-blu della popolazione 32Dkit </li></ul></li><li> Utilizzare Microsoft Excel per rappresentare graficamente la fluorescenza media (Figura 2a). Calcolare la pendenza della linea tra i 5 ei 10 minuti. Grafico la pendenza come un grafico a barre per determinare l&#39;attività calpaina nelle cellule (Figura 2b). </li></ol><img src="/files/ftp_upload/2050/2050fig1a.jpg" alt="Figura 1a" /> Figura 1a. Determinazione cellule vive in base al profilo scatter in avanti e laterale. <img src="/files/ftp_upload/2050/2050fig1b.jpg" alt="Figura 1b" /> Figura 1b. Variazione media fluorescenza nel corso di 10 minuti di tempo. <img src="/files/ftp_upload/2050/2050fig2a.jpg" alt="Figura 2a" /> Figura 2a. Rappresentante grafico di fluorescenza media. <img src="/files/ftp_upload/2050/2050fig2b.jpg" alt="Figura 2b" /> Figura 2b. Calpain attività nelle cellule 32Dkit con inibitori.

<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>

Nessun conflitto di interessi dichiarati.

L&#39;attività calpaina in cellule 32Dkit fisse e vita è stato determinato in questo protocollo video. Il trattamento della linea cellulare con l&#39;inibitore della calpaina PD150606 portato a completa inibizione dell&#39;attività calpaina nelle cellule. Inoltre, il trattamento con l&#39;inibitore MEK1 PD98059 portato a completa inibizione dell&#39;attività calpaina nelle cellule. ERK è un importante attivatore di calpaina-2 3. Questi risultati suggeriscono che l&#39;attività calpaina-2 è predominante...

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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>
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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

Calpains sono onnipresenti intracellulari, calcio-sensibili, la cisteina proteasi neutre<sup&gt; 1</sup&gt;. Calpains svolgono un ruolo cruciale in molti processi fisiologici, tra cui segnalazione, rimodellamento del citoscheletro, regolazione dell&#39;espressione genica, l&#39;apoptosi e la progressione del ciclo cellulare<sup&gt; 1</sup&gt;. Calpains sono stati coinvolti in molte patologie tra cui le distrofie muscolari, cancro, diabete, morbo di Alzheimer e la sclerosi multipla<sup&gt; 1</sup&gt;. Calpaina regolamento è complesso e non completamente compreso. livelli di mRNA e proteine ​​correlate con l&#39;attività poco, limitando l&#39;uso di tecniche di espressione dei geni o proteine ​​per misurare l&#39;attività calpaina. Questo il video dettagli protocollo un test citometria a flusso sviluppato nel nostro laboratorio per la misurazione dell&#39;attività calpaina in cellule fisse e viventi. Questo metodo utilizza il substrato fluorescente BOC-LM-CMAC, che viene scissa in particolare da calpaina, per misurare l&#39;attività calpaina.<sup&gt; 2</sup&gt; In questo video, attività calpaina nelle cellule 32Dkit fisso e vive leucemia murina, da soli o come parte di una popolazione splenocyte viene misurata utilizzando un LSRII (BD Bioscience). Le cellule sono 32Dkit dimostrato di avere attività elevata rispetto a splenociti normali.

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

Questo articolo dettaglio il protocollo per la misurazione dell&#39;attività calpaina in cellule fisse e vivere in citometria a flusso.

Misurazione dell&#39;attività Calpain nelle celle fissi e Living in citometria a flusso

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 Views.  University of Toronto. Questo articolo dettaglio il protocollo per la misurazione dell'attività calpaina in cellule fisse e vivere in citometria a flusso.

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.

Umano In Vitro</emSoppressione> come strumento di screening per il riconoscimento di uno Stato precoce di squilibrio immunitario

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

Ricerca

Immunologia e infezioni

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 seguito di Cell-destino<em&gt; E. coli</em&gt; Dopo l&#39;infezione da 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.

Esame di Selezione timica positivi e negativi per Citometria a Flusso

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.

Misurazione Attaccamento e internalizzazione del virus A in cellule A549 mediante citometria di flusso

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.

Separazione delle cellule fluorescenza-attivato per la purificazione delle cellule dendritiche plasmacitoidi dal midollo osseo di topo

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 coltura In Vitro dei macrofagi alveolari murini ed umani

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.

Preparazione del midollo osseo intero per l'analisi della citometria di massa delle cellule di lignaggio neutrofilo

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.

Misurare la fagocitosi di Aspergillus fumigatus Conidia da parte dei Leucociti Umani utilizzando Flow Cytometry

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).

Misurazione del recettore del complemento dell'eritrocite 1 utilizzando la citometria di flusso

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

Misurazione dell'attività Calpain nelle celle fissi e Living in citometria a flusso

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