Unterstützt durch Zuschüsse aus dem Canadian Institutes of Health Research, The Leukemia and Lymphoma Society of Canada und der Lymphoma Foundation of Canada.

Bereiten Sie Reagenzien Hinweis: Alle PBS enthält Ca 2 + und Mg 2 + <ol><li> Nachweisreagenz Add 20 um 7-Amino-4-chlormethyl Cumarin, t-BOC-Leucin-methioninamid (BOC-LM-CMAC) auf Raumtemperatur PBS. Jede Zellsuspension erfordert 2mL Nachweisreagenz. </li><li> 1% Paraformaldehyd (Fixed Cells) Verdünnen Sie 4% Paraformaldehyd Stammlösung bei Raumtemperatur PBS. Aliquot 1ml 1% Paraformaldehyd jedem FACS-Röhrchen. Drei FACS Röhrchen für jede experimentelle Bedingung erforderlich sind (zB 32Dkit, 32Dkit + PD150606, 32Dkit + PD98059 brauchen 9 FACS-Röhrchen) </li><li> PD150606 (Calpain-Inhibitor) Rekonstituieren PD150606 in DMSO in einer Endkonzentration von 100 mM. Schützen Sie dieses Reagenz und alle Zellen mit Inhalt vor Licht behandelt für die Dauer des Experiments. </li><li> PD98059 (MEK1 Inhibitor) Rekonstituieren PD98059 in DMSO in einer Endkonzentration von 50mM. </li></ol> Bereiten Cells <ol><li> Wachsen 32Dkit Zellen bei 5 x 05 bis 1 Oktober x 10 6 Zellen pro ml in Opti-MEM Medium mit 5% fötalem Rinderserum, WEHI-3 Überstand als eine Quelle von IL-3 (oder eine andere Quelle für IL-3, wie ergänzt als rekombinantes Protein), 0,1% 2-Mercaptoethanol und Antibiotika. </li><li> Sammeln Sie 12 x 10 6 Zellen 32Dkit in drei 15 mL Falcon-Röhrchen (4 x 10 6 Zellen pro Röhrchen). </li><li> Pellet die Zellen bei 1000 rpm für 5 Minuten bei 15 ° C. </li><li> Den Überstand aspirieren. Resuspendieren der Zellen in 2 ml Raumtemperatur PBS. </li><li> Gönnen einer Zelle Fahrwerk mit 50 &amp;mgr; PD150606. Sicherstellen, dass die Probe von Licht durch das Abdecken der Falcon-Röhrchen mit Alu-Folie geschützt. Vortex kurz danach für 20-30 Minuten bei Raumtemperatur im Dunkeln inkubieren. </li><li> Treat der zweiten Zellsuspension mit 20 um PD98059. Platte die Zellen in einer 35-mm Gewebe Kulturschale und Inkubation für 2 Stunden bei 37 ° C. </li></ol> Calpain Assay in fixierten Zellen <ol><li> Während der Proben mit PD150606 sind Inkubation beginnen die Calpain-Assay auf den unbehandelten Proben. Beginnen Sie, indem 2 ml Nachweisreagenz jeder Zellsuspension. Unmittelbar 1ml der Zellsuspension / Nachweisreagenz Mischung auf die vorbereiteten FACS-Röhrchen mit 1% Paraformaldehyd (0 Minuten Zeitpunkt). </li><li> Inkubieren Zellsuspensionen mit Nachweisreagenz für 5 Minuten bei Raumtemperatur. Dann 1ml der Zellsuspension zu einem FACS-Röhrchen mit 1 ml 1% Paraformaldehyd. </li><li> Weiter Inkubation der Zellsuspensionen mit Erkennung Mischung für weitere 5 Minuten bei Raumtemperatur. Dann 1ml der Zellsuspension zu einem FACS-Röhrchen mit 1 ml 1% Paraformaldehyd. </li><li> Wiederholen Sie die Calpain-Assay auf 32Dkit Zellen, die mit Inhibitoren behandelt wurden. </li><li> Fix Zellen über Nacht im Dunkeln bei 4 ° C. </li><li> Am nächsten Tag Pellet die Zellen bei 1800 rpm für 5 Minuten bei 15 ° C. </li><li> Den Überstand aspirieren. Resuspendieren der Zellen in 750uL PBS. Legen Zellen auf Eis und halten in der Dunkelheit. </li></ol> Erfassung von Daten für die fixierten Zellen <ol><li> Analysieren Sie die Zellen mit einer LSR II (BD Bioscience). Die Filter sind für die Lightwave Xcyte (UV)-Laser, Anregung Linie 355nm und Laserleistung 25mW, die Emission von 405 und 450 nm für Substrat und Produkt bzw. eingestellt. Bandpass-Filter für Substrat und Produkt sind 405/20 und 450/50. Langpassfilter für Substrat und Produkt sind 405 und 450. Die PMT-Spannung wurde auf 350-375 für Substrat und 325-350 für das Produkt eingestellt. </li><li> Set-up eine BD FACSDiva Experiment mit den folgenden Parametern: forward scatter; side scatter; Indo-1 Blue (log); Indo-1 Violet (log). Auf dem Arbeitsblatt einrichten die folgenden Grafiken: forward scatter vs side scatter Dot-Plot mit einem Tor an lebenden Zellen (Abbildung 1a), Indo-1 Blue-Histogramm, Indo-1 Violet Histogramm, Indo-1 Blue vs Indo-1 Violet Dot-Plot. </li><li> Kalibrieren Sie den LSR II mit AlignFlow und AlignFlow sowie fluoreszierende Kügelchen (Invitrogen). Die PMT-Spannung soll an die Spitze des Bead-Fluoreszenz-Histogramm in dem gleichen Kanal vor jedem Experiment Ort sein. </li><li> Begin Erfassung von Daten über die 32Dkit Zellen in der 0-Minuten-Zeitpunkt. Stellen Sie Parameter Spannungen wie nötig. Erfassen und speichern, 10.000 Ereignisse pro Probe. </li><li> Wiederholen Sie die Übernahme für jede Probe, Spülen der Maschine mit FACS-Puffer zwischen den einzelnen Röhre. </li><li> Export der Daten. Stellen Sie sicher, LSR II ist nach Instituts-Richtlinien gereinigt. </li></ol> Calpain Assay in lebenden Zellen <ol><li> Bereiten Sie die Zellsuspensionen wie oben, ohne PD150606. Bringen Sie die Proben an die LSR II und Filter einrichten und Parameter wie oben. Richten Sie das Arbeitsblatt wie oben. Fügen Sie eine große Dot-Plot zeigt Indo-1 Blue-Zeit. Stellen Sie das Programm, um die maximale Zahl erwerbenber von Veranstaltungen (kein Stop-Tor). </li><li> Add 50 &amp;mgr; PD150606 zu einer Zellsuspension von 32Dkit Zellen. Halten Sie die Probe im Dunkeln und bei Raumtemperatur inkubieren für 20-30 Minuten. </li><li> Begin Erfassung von Daten über die 32Dkit Zellsuspension ohne PD150606. Verwenden Sie eine geringe Strömungsgeschwindigkeit von 200-300 Ereignisse pro Sekunde. Nach 30 Sekunden bis 1 Minute den Schlauch aus der Maschine. Add 20 um BOC-LM-CMAC zu den Zellen. Vortex kurz danach weiterhin den Erwerb der Daten. </li><li> Erfassung von Daten für 10-20 Minuten oder bis der Calpain-Aktivität Plateaus. </li><li> Wiederholen Sie die Schritte 3 und 4 mit 32Dkit Zellen mit PD150606 behandelt. </li><li> Export der Daten. Stellen Sie sicher, LSR II ist nach Instituts-Richtlinien gereinigt. </li></ol> Calpain Assay in Complex (Mixed) Zellpopulationen * Dieses Experiment wird 32Dkit Zellen in einer Zellsuspension aus einer Maus-Milz geerntet zu identifizieren. <ol><li> Ernte der Milz von jeder Maus. Lyse der roten Blutkörperchen mit NH 4 Cl für 10 Minuten bei Raumtemperatur. </li><li> Bereiten Sie einzelne Zellsuspensionen in 2 ml PBS. </li><li> Teilen Sie jede Zellsuspension in zwei Teile. Gönnen 1 Zelle Fahrwerk mit 50 &amp;mgr; PD150606 für 20-30 Minuten bei Raumtemperatur im Dunkeln. </li><li> Gehen Sie wie oben für die Calpain Assay in fixierten Zellen (Schritte 1-6) </li><li> Den Überstand aspirieren. Waschen Sie die Zellen in 500μL PBS. Den Überstand aspirieren. </li><li> Resuspendieren der Zellen in 200 ul Färbepuffer mit 2% BSA. Inkubieren für 15 Minuten bei Raumtemperatur. </li><li> Bereiten Sie den primären Antikörper (FITC-anti-Maus CD117). Der Antikörper sollte bei einer Verdünnung von 1:200 in Färbepuffer verwendet werden. Sicherstellen, dass die Antikörper im Dunkeln gehalten wird. </li><li> Add 200 ul primären Antikörpers zu den Zellen. Inkubieren für 30 Minuten bei Raumtemperatur im Dunkeln. </li><li> Fügen Sie 400μL PBS, um die Lautstärke für die Durchflusszytometrie erhöhen. Legen Sie die Zellen auf Eis und halten in der Dunkelheit. </li><li> Gehen Sie wie in Acquire Daten für fixierte Zellen beschrieben. Add FITC, um die Parameter und verfügen über ein Histogramm für FITC auf dem Arbeitsblatt. </li></ol> Analyse und repräsentative Ergebnisse <ol><li> Analysieren von Daten mit FlowJo (Treestar). Tor an lebenden Zellen auf Vorwärts-und Seitwärts-Streulicht-Profil (Abb. 1a) basiert. Bestimmen Sie die mittlere Fluoreszenz für Indo-Blue aus der lebensfähigen Zellen Tor (Abb. 1b). </li><li> a) Für die gemischten Zellpopulation <ul><li> Verwendung FITC Fluoreszenz-Histogramme und Dot-Plots der 32Dkit Bevölkerung identifizieren </li><li> Bestimmen Sie die mittlere Fluoreszenz für Indo-Blue der 32Dkit Bevölkerung </li></ul></li><li> Verwenden Sie Microsoft Excel, um Graphen die mittlere Fluoreszenz (Abbildung 2a). Berechnen Sie die Steigung der Geraden zwischen 5 und 10 Minuten. Graph die Steigung als Balkendiagramm an Calpain-Aktivität in den Zellen zu bestimmen (Abbildung 2b). </li></ol><img src="/files/ftp_upload/2050/2050fig1a.jpg" alt="Abbildung 1a" /> Abbildung 1a. Bestimmung lebenden Zellen auf Vorwärts-und Seitwärts-Streulicht-Profil. <img src="/files/ftp_upload/2050/2050fig1b.jpg" alt="Abbildung 1b" /> Abbildung 1b. Mittlere Fluoreszenz Wechsler 10 Minuten Zeit, natürlich. <img src="/files/ftp_upload/2050/2050fig2a.jpg" alt="Abbildung 2a" /> Abbildung 2a. Representative Graph des durchschnittlichen Fluoreszenz. <img src="/files/ftp_upload/2050/2050fig2b.jpg" alt="Abbildung 2b" /> Abbildung 2b. Calpain-Aktivität in 32Dkit Zellen mit Inhibitoren.

<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>

Die Calpain-Aktivität in fixierten und lebenden Zellen 32Dkit hat in diesem Video-Protokoll bestimmt. Die Behandlung der Zelllinie mit dem Calpain-Inhibitor PD150606 führte zu einer vollständigen Hemmung von Calpain-Aktivität in den Zellen. Darüber hinaus führte die Behandlung mit dem Inhibitor PD98059 MEK1 in eine vollständige Hemmung von Calpain-Aktivität in den Zellen. ERK ist ein wichtiger Aktivator von Calpain-2 3. Diese Ergebnisse legen nahe, dass Calpain-2-Aktivität überwiegt in der 32Dkit Zellen ist. Aktuelle Arbeiten in unserem Labor untersucht die Folgen der erhöhten Calpain-Aktivität und die Beiträge von Calpain-1 und Calpain-2 bei Leukämie. Dieses Protokoll Details der ersten durchflusszytometrischen basierender Assay zur Calpain Aktivität in fixierten und lebenden Zellen zu messen und kann verwendet werden, um den Mechanismus von Calpain Regulierung und ihre Rolle in pathologischen Prozessen mit intakten Zellen zu verstehen. Dieses Protokoll kann geändert werden, um die Auswirkungen von Inhibitoren oder Gen-Manipulationen an Calpain-Aktivität zu untersuchen. Darüber hinaus kann dieses Tests verwendet werden, um Calpain-Aktivität in einer komplexen Mischung von Zellen, wie zB Nabelschnurblut oder zu identifizieren und zu messen Calpain-Aktivität in Zell-Subpopulationen aus der Milz isoliert zu messen. Dieser Test unterscheidet nicht zwischen Aktivität durch verschiedene Isoformen von Calpain erzeugten unterscheiden. Genetische oder pharmakologische Reagenzien, die Knock-down oder hemmen bestimmte Calpainen kann verwendet werden, um dieses Problem zu beheben sein.

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		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>PBS BOC-LM-CMAC 4% paraformaldehyde</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td> Dissolve 4g paraformaldehyde in 100mL PBS. In fume hood, heat sample with stirring until clear. Let cool then store at 4&deg;C</td>
</tr>
<tr>
<td>PD150606</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>Use freshly reconstituted PD150606 for maximal calpain inhibition.</td>
</tr>
<tr>
<td>PD98059</td>
<td>&nbsp;</td>
<td>Cell Signaling Technology</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>AlignFlow, AlignFlow plus fluorescent beads FACS tubes</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>LSR II</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>&nbsp;</td>
<td> Supernatant of WEHI-3 cells is used as a source of IL-3 in these experiments. Each collection of WEHI-3 supernatant is titrated to determine the optimal concentration used for 32Dkit cell growth. </td>
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measuring calpain activity in fixed and living cells by flow cytometry

Calpaine sind allgegenwärtig intrazellulären Calcium-sensitiven, neutral Cystein-Proteasen<sup&gt; 1</sup&gt;. Calpaine spielen eine entscheidende Rolle in vielen physiologischen Prozessen, einschließlich der Signalisierung, Zytoskelett-Umbau, Regulation der Genexpression, Apoptose und Zellzyklus-Progression<sup&gt; 1</sup&gt;. Calpaine haben in vielen Pathologien wie Muskeldystrophien, Krebs, Diabetes, Alzheimer und Multipler Sklerose beteiligt<sup&gt; 1</sup&gt;. Calpain Regelung ist komplex und noch nicht vollständig geklärt. mRNA-und Proteinebene korrelieren schlecht mit der Tätigkeit, Beschränkung der Verwendung von Gen-oder Protein-Expression Techniken Calpain-Aktivität zu messen. Dieses Video-Protokoll Details ein durchflusszytometrischen Assays in unserem Labor zur Messung von Calpain-Aktivität in fixierten und lebenden Zellen entwickelt. Diese Methode verwendet das fluoreszierende Substrat BOC-LM-CMAC, die speziell durch Calpain gespalten, um Calpain-Aktivität zu messen.<sup&gt; 2</sup&gt; In diesem Video wird Calpain-Aktivität in fixierten und lebenden Maus 32Dkit Leukämiezellen, allein oder als Teil eines Splenozyten Bevölkerung gemessen mit einem LSRII (BD Bioscience). 32Dkit Zellen gezeigt zu haben, erhöhte Aktivität in den normalen Splenozyten verglichen.

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

Dieser Artikel wird detailliert die Verfahren zur Messung der Calpain-Aktivität in fixierten und lebenden Zellen mittels Durchflusszytometrie.

Mess-Calpain Aktivität in fixierten und lebenden Zellen mittels Durchflusszytometrie

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 Aufrufe.  University of Toronto. Dieser Artikel wird detailliert die Verfahren zur Messung der Calpain-Aktivität in fixierten und lebenden Zellen mittels Durchflusszytometrie.

Serie: 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.

Mensch In Vitro Suppression als Screening-Tool für die Anerkennung von 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 ...

Forschung

Immunologie und Infektion

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.

Nach Cell-Schicksal<em&gt; E. coli</em&gt; Nach Infektion mit Phagen

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.

Prüfung Thymic positive und negative Selektion mittels Durchflusszytometrie

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.

Mess Bindung und Internalisierung von Influenza A-Virus in A549-Zellen mittels Durchflusszytometrie

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.

Fluoreszenz-aktivierte Zellsortierung zur Reinigung von Plasmazytoide dendritischer Zellen aus dem Knochenmark der Maus

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.

Isolation und In-vitro- Kultur von murinen und menschlichen alveoläre Makrophagen

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.

Vorbereitung des ganzen Knochenmarks für die Mass Cytometry Analyse von Neutrophilen-Linienzellen

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.

Messung der Phagozytose von Aspergillus fumigatus Conidia durch menschliche Leukozyten mit Flow Cytometrie

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).

Messung des Erythrozytenkomplementrezeptors 1 mit Durchflusszytometrie

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