Der Autor bedankt sich bei Frau Lora Matthews für die Durchführung der Technik und Herr Paul Shepherd für Kinematographie danken. Dieses Projekt wurde von der University of South Australia, Laboratoriumsmedizin Programm gefördert.

Vorbereitung von 5x Veronal Buffered Saline (VBS) <ol><li> Zur Vorbereitung der Veronal-gepufferte Kochsalzlösung (VBS), müssen drei separate Lösungen vorbereitet werden. </li><li> Bereiten Sie die Lösung 1 durch Auflösen 21.25gm von NaCl und 0.94gm von Sodium barbiturathaltiges Mittel in 350ml destilliertem Wasser. Die Endkonzentrationen von NaCl und Natrium barbiturathaltiges Mittel sind 1,02 M und 13mm bzw.. </li><li> Bereiten Sie die Lösung 2 durch Lösen 1.44gm von barbiturathaltiges Mittel in 125ml destilliertem Wasser heiß. Die Endkonzentration der barbiturathaltiges Mittel ist 62.5mm. </li><li> Bereiten Lösung 3 durch Auflösen 20.33gm von MgCl 2 und 4.41gm von CaCl 2 in 100 ml destilliertem Wasser. Die Endkonzentration von MgCl 2 und CaCl 2 ist 2,18 m und 440 jeweils. </li><li> Mix-Lösungen 1 und 2 und auf Raumtemperatur abkühlen. </li><li> Sobald die kombinierte Lösung abgekühlt ist, fügen Sie 1,25 ml der Lösung 3 und pH-Wert auf 7,3-7,5 mit 1 M HCl. </li><li> Passen Sie das endgültige Volumen auf 500 ml mit destilliertem Wasser auf einem 5-fach Stammlösung. </li><li> So bereiten Sie eine 1x funktionierende Lösung, verdünnte die Aktie im Verhältnis 1:5 mit destilliertem Wasser. </li></ol> Sensibilisierung von Schafs-Erythrozyten mit Hämolysin <ol><li> Bereiten Sie das Hämolysin, indem zunächst Verdünnung 1:50 mit VBS </li><li> Um 4 ml SRBC hinzufügen 6ml des VBS und vorsichtig durch Umdrehen mischen </li><li> Zentrifugation bei 600g x 5 Minuten </li><li> Überstand verwerfen und die Zellen weitere 2 Mal </li><li> Nach dem letzten Waschen Zentrifuge die Zellen bei 900g x 5 Minuten, um die Zellen-Pack </li><li> Überstand verwerfen und Zellen in ausreichender VBS zu einer 10% igen Lösung, dh 0,5 ml gepackte Zellen herzustellen, sind in 5 ml Puffer </li><li> Tropfenweise das gleiche Volumen von Hämolysin (Kaninchen Anti-Schaf-Erythrozyten-Antikörper), um die Zellen während wirbelnde kontinuierlich </li><li> Inkubation bei 30 ° C für 30 Minuten in einem Wasserbad </li><li> Vorsichtig mischen die Zellen alle 15 Minuten </li><li> Sensibilisiert SRBC kann über Nacht bei 4 ° C gelagert werden </li></ol> CH 50-Test <ol><li> Label eine Reihe von Röhren in zweifacher Ausfertigung mit 1:8, 1:16, 1:32, 1:64 und 1:128. </li><li> Bereiten Sie eine Reihe von zwei fache serielle Verdünnungen von Kontroll-und Test-Serum in VBS jeweils im Doppel </li><li> Start bei 1:4 (100ml Serum + 300ml VBS) und Transfer 200ml Probe in den nächsten markierten Röhre. </li><li> Nach gründlichem Mischen zwischen Verdünnungen und Transfer 200ml zum nächsten Verdünnung mit einer frischen Pipettenspitze. </li><li> Wiederholen, bis alle fünf Verdünnungen hergestellt werden. </li><li> Discard 200ml von der endgültigen 1:128 Verdünnung. </li><li> Add 200 ml suspendiert sensibilisiert SRBC allen Rohren. </li><li> Label zwei getrennten Röhren als BLANK und fügen 200ml sensibilisierter SRBC + 200ml VBS. Diese Rohre messen spontane Lyse der SRBC im VBS. </li><li> Label anderen zwei getrennten Röhren als TOTAL Lyse und fügen Sie 200 ml sensibilisiert SRBC + 200ml destilliertem Wasser. </li><li> Vorsichtig mischen allen Rohren. </li><li> Bei 37 ° C für 30 Minuten in einem Wasserbad Mischen nach 15 Minuten. </li><li> Zentrifugieren Sie die Proben bei 1.500 g für 5 Minuten in das Sediment des RBC. </li><li> Transfer 100ml Überstand aus jeder Röhre ein gut in einer 96-well ebenen Bodenplatte. </li><li> Add 100 ml destilliertem Wasser in jede Vertiefung. </li><li> Lesen Sie die Absorption der Proben bei 540 nm mit einer Platte Spektralphotometer. </li></ol> Berechnungen <ol><li> Berechnen Sie die mittlere Absorption für jede Probe </li><li> Subtrahieren Sie die BLANK Absorption (spontane Lyse) von allen Proben </li><li> Berechnen Sie den% Lyse für jede Verdünnung mit folgender Formel: <img src="/files/ftp_upload/1923/1923eq1.jpg" alt="Gleichung" /></li><li> Zeichnen Sie die prozentuale Lyse (vertikale Achse) gegen die Serumverdünnung auf der horizontalen Achse. </li><li> Berechnen Sie die Verwässerung für die 50% ige Hämolyse für die Steuerung und Testserum (Abb. 2) benötigt. </li></ol> Repräsentative Ergebnisse: Das Video enthält ein Beispiel für repräsentative Ergebnisse. In der Praxis wird ein Kontrollserum auch zur gleichen Zeit wie die Testserum geführt und ist in der gleichen Weise behandelt. Below (Tabelle 1) Sample-Daten aus einer getesteten Serumprobe. Die Daten werden nach der Gleichung in 5.3 vorgestellt manipuliert. <table border="1"><tbody><tr><td> Probe </td><td> OD 540 </td><td> Bedeuten </td><td></td><td></td></tr><tr><td> Leer </td><td> 0,042, 0,044 </td><td> 0,043 </td><td></td><td></td></tr><tr><td> Insgesamt Lyse </td><td> 0,183, 0,183 </td><td> 0,183 </td><td></td><td></td></tr><tr><td colspan="5"></td></tr><tr><td> Verdünnung </td><td> OD 540 </td><td> Bedeuten </td><td> Mean-Blank </td><td> % Lysis </td></tr><tr><td> 01.08 </td><td> 0,200, 0,219 </td><td> 0,210 </td><td> 0,168 </td><td> 116 </td></tr><tr><td> 01.16 </td><td> 0,179, 0,173 </td><td> 0,176 </td><td> 0,134 </td><td> 93 </td></tr><tr><td> 01.32 </td><td> 0,134, 0,110 </td><td> 0,122 </td><td> 0,08 </td><td> 55 </td></tr><tr><td> 1:64 </td><td> 0,053, 0,066 </td><td> 0,059 </td><td> 0,017 </td><td> 12 </td></tr><tr><td> 1:128 </td><td> 0,044, 0,045 </td><td> 0,045 </td><td> 0,003 </td><td> 2 </td></tr></tbody></table><img src="http://www.jove.com/files/ftp_upload/1923/1923fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Die Aktivierung des klassischen Komplementweges. Der klassische Weg wird durch die Bindung Immunglobulin-M (IgM) oder Immunglobulin-G (IgG) auf der Oberfläche einer Zielzelle aktiviert. Der Fc-Teil des Ab bindet C1q, ist C1r aktiviert und dies wiederum aktiviert ein anderes Molekül C1r die zusammen aktivieren zwei Moleküle C1s. C1s spaltet C4 nun die Bindungsstelle für C2, die ebenfalls gespalten wird entlarvt. Die Bindung von C4b und C2a führt zur Bildung eines Komplexes bezeichnet als C3-Konvertase. Dieser Komplex jetzt spaltet C3 bildet C3a und C3b, von denen einige mit dem C3-Konvertase Bildung einer C5 Konvertase kombinieren. Dieser Komplex wirkt nun auf C5, mit den daraus resultierenden C5b Bindung an C6 initiieren die Bildung der Membran-Angriffs-Komplex (MAC). C5b6 wirkt auf C7, was wiederum wirken auf C8 und letztlich auf C9, was zur Bildung des endgültigen MAC. (Goldsby et al, 2003). <img src="http://www.jove.com/files/ftp_upload/1923/1923fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Plotten von Sample-Daten und die Berechnung der CH50 für eine Steuer-und Testserumprobe. (A), sind die berechneten Prozentsatz (%) Lyse sowohl der Kontrollgruppe (•) und test () Serumproben gegen Verdünnungsfaktor aufgetragen. (B) Für die Berechnung der 50%-Lyse ist eine Linie von der 50% Prozent-Wert gezogen, bis sie mit dem Graphen Geraden schneidet und dann eine senkrechte Linie nach unten, um die Verdünnung gezogen. In diesem Beispiel werden ein 35-facher Verdünnung der Kontroll-und 21,6-fachen Verdünnung des Testserum verpflichtet, 50% Lyse zu erreichen. Diese Daten deuten darauf hin, dass es weniger Ergänzung im Testserum verglichen mit der Kontrollgruppe, da sie weniger Verdünnung erforderlich, bevor 50% Lyse wurde erreicht. Die Kontrollprobe zeigt auch&gt; 100% Lyse bei der 1:8 Verdünnung. Dies ist wahrscheinlich aufgrund von unvollständigen Lyse der SRBC durch das destillierte Wasser und effizientere Lyse durch den Komplement-Komponenten.

<ol>
	<li>Goldsby, R. A., Kindt, T. J., Osborne, B. A., Kuby, 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> Immunology. , (2003).</li></ol>

Die CH 50-Test unterliegt vielen Störungen. Einige SRBC sind empfindlicher als andere, was zu spontanen Hämolyse, die nicht mit Aktivitäten zu ergänzen ist. Die Affinität der Kaninchen-Antikörper variiert von Charge zu Charge und von einem Hersteller zum anderen, dies beeinflusst die Höhe des Antikörper, der an der SRBC bindet. Auch der Prozess der Sensibilisierung SRBC mit Antikörper Ergebnisse in Zellen mit unterschiedlichen Mengen der Antikörper-Beschichtung der SRBC. Gewinnung und Lagerung sind ...

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<td>Sheep red blood cells</td>
<td>&nbsp;</td>
<td>CSL</td>
<td>2490201</td>
<td>Use at 1% final</td>
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<td>Serum</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Human serum</td>
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<td>Rabbit Anti-Sheep Haemolytic serum (RBCs), Unconjugated</td>
<td>&nbsp;</td>
<td>AdB Serotec</td>
<td>C12HSB</td>
<td>&nbsp;</td>
<tr>
<td>96 well flat bottom plate</td>
<td>&nbsp;</td>
<td>Sarstedt</td>
<td>83.1839</td>
<td>&nbsp;</td>
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<td>Veronal buffered saline</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Use at 1x final</td>
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<td>Waterbath</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>37&deg;C room/incubator</td>
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measuring the 50% haemolytic complement (ch50) activity of serum

Das Komplementsystem ist eine Gruppe von Proteinen, die bei Aktivierung führen zu Zelllyse Ziel und erleichtert die Phagozytose durch Opsonisierung. Individuelle Komplement-Komponenten quantifiziert Dies bedeutet jedoch keine Auskunft über die Aktivität der Weg sein. Die CH<sub&gt; 50</sub&gt; Ist ein Screening-Test für die Aktivierung des klassischen Komplementweges (Abb. 1) und es ist empfindlich auf die Reduktion, Abwesenheit und / oder Untätigkeit eines jeden Teils des Weges. Die CH<sub&gt; 50</sub&gt; Tests die Funktionsfähigkeit des Serum-Komplement-Komponenten von den klassischen Weg, um rote Blutzellen von Schafen (SRBC) vorbeschichtet mit Kaninchen-Anti-Schaf-Erythrozyten-Antikörper (Hämolysin) zu lysieren. Als Antikörper-beschichteten SRBC mit Testserum, den klassischen Weg der Komplementaktivierung wird aktiviert und Hämolyse Ergebnisse inkubiert. Wenn ein Komplement-Komponente fehlt, wird der CH<sub&gt; 50</sub&gt; Ebene Null sein wird, wenn eine oder mehrere Komponenten des klassischen Weges sind zurückgegangen, die CH<sub&gt; 50</sub&gt; Verringert. Ein festes Volumen von optimal sensibilisiert SRBC wird jeder Serumverdünnung aufgenommen. Nach der Inkubation wird das Gemisch zentrifugiert und der Grad der Hämolyse wird durch Messung der Absorption des Hämoglobins in den Überstand bei 540 nm freigesetzt quantifiziert. Die Höhe der Komplement-Aktivität wird durch die Untersuchung die Fähigkeit der verschiedenen Verdünnungen der Test Serum-Antikörper beschichtet SRBC lysieren bestimmt. Dieses Video beschreibt die experimentellen Schritte bei der Analyse der Ebene der Komplement-Aktivität des klassischen Komplementweges beteiligt.

Watch this Scientific Journal Video about Measuring the 50% Haemolytic Complement CH50 Activity of Serum at JoVE.com

Measuring the 50% Haemolytic Complement CH50 Activity of Serum

Der klassische Weg wird durch Antikörper aktiviert und gipfelt in Ziel-Zell-Lyse. Die CH<sub&gt; 50</sub&gt; Test liefert ein Maß für die Komplement-Aktivität einer Serumprobe. Dieses Video zeigt die Schritte bei der Bestimmung der CH beteiligt<sub&gt; 50</sub&gt; Einer Serumprobe, die Berechnungen und Interpretation der Ergebnisse.

Die Messung der 50% Hämolytische Complement (CH 50) Aktivität von Serum

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual ...

measuring-the-50-haemolytic-complement-ch50-activity-of-serum

Research

JoVE Journal

Immunology and Infection

Measuring the 50% Haemolytic Complement (CH50) Activity of Serum

37.5K Aufrufe.  University of South Australia. Der klassische Weg wird durch Antikörper aktiviert und gipfelt in Ziel-Zell-Lyse. Die CH 50 Test liefert ein Maß für die Komplement-Aktivität einer Serumprobe. Dieses Video zeigt die Schritte bei der Bestimmung der CH beteiligt 50 Einer Serumprobe, die Berechnungen und Interpretation der Ergebnisse.

Serie: Measuring the 50% Haemolytic Complement CH50 Activity of Serum

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

Depletion spezifischer Zellpopulationen durch Ergänzung Depletion

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

Forschung

Immunologie und Infektion

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood cells are particularly useful targets as they are readily available and agglutination is observable using the naked eye. This technique is commonly used to determine the titre of an antibody (Ab), for blood grouping and viral quantification. In this video, the steps involved in preparing and performing a haemagglutination assay is demonstrated using antibodies specific to blood group A-antigens added to red blood cells (Revercells). The antiserum is serially diluted in a 96 well U-bottom microtitre tray, to which is added a suspension of Revercells. The samples are mixed and then incubated at 37&deg;C for 60 minutes. After this time, the samples can then be easily scored for  ve, +ve and intermediate (-/+) haemagglutination reactions. This approach allows for the reactivity and titre of a serum sample to be assessed using a rapid and simple technique. The video will cover the theory behind the assay, how the results are read and interpreted, how the titre is determined, how the assay can be modified and any issues associated with the use of this technique.

Haemagglutination is a form of agglutination where antibodies bind to red blood cells. Red blood cells are both readily available and the results are readily observable using the naked eye. This video demonstrates the steps involved in a haemagglutination assay, interpreting the results and determining the titre.

Die Bestimmung der Reaktivität und Titre von Serum mit einem Hämagglutinations Assay

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood ...

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

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 Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Mess-bakterielle Belastung und Immunreaktionen in Mäuse Infizierte mit Listeria monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

Die Bestimmung der Phagozytoseaktivität of Clinical Antikörper Proben

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Speichel, Speicheldrüse, und Hämolymphe Abholung Ixodes scapularis Zecken

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

Methoden für die quantitative Bestimmung von Antikörper-induzierte Komplementaktivierung auf Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

Mess Lokale Anaphylaxie bei Mäusen

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

Mess Granulozyten und Monozyten Phagozytose und oxidativer Burst-Aktivität im menschlichen Blut

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin acquisition at the bacterial cell surface plays in resistance to the host immune system. Flow cytometry experiments identified human plasma vitronectin as a ligand for the bacterial receptor outer membrane protein H of Haemophilus influenzae type f. An enzyme-linked immunosorbent assay was employed to characterize the protein-protein interactions between purified recombinant protein H and vitronectin, and binding affinity was assessed using bio-layer interferometry. The biological importance of the binding of vitronectin to protein H at the bacterial cell surface in evasion of the host immune response was confirmed using a serum resistance assay with normal and vitronectin-depleted human serum. The importance of vitronectin in bacterial adherence was analyzed using glass slides with and without vitronectin coating, followed by Gram staining. Finally, bacterial adhesion to human alveolar epithelial cell monolayers was investigated. The protocols described here can be easily adapted to the study of any bacterial species of interest.

This report describes protocols for characterizing interactions between bacterial outer membrane proteins and the human complement regulator vitronectin. The protocols can be used to study the binding reactions and biological function of vitronectin in any bacterial species.

Assays für die Untersuchung der Rolle von Vitronectin in bakterielle Adhäsion und Serum-Widerstand

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin ...