L&#39;auteur tient à remercier Mlle Lora Matthews pour l&#39;exécution de la technique et de M. Paul Berger pour la cinématographie. Ce projet a été financé par l&#39;Université d&#39;Australie du Sud, le programme de médecine de laboratoire.

Préparation de la solution saline tamponnée 5x véronal (VBS) <ol><li> Pour préparer la Saline véronal Buffered (VBS), trois solutions distinctes doivent être préparées. </li><li> Préparer la solution en dissolvant une 21.25gm de NaCl et d&#39;0.94gm barbitone de sodium dans 350ml d&#39;eau distillée. Les concentrations finales de NaCl et de sodium sont barbitone 1,02 et 13mm respectivement. </li><li> Préparer la solution 2 en dissolvant 1.44gm des barbitone dans 125ml d&#39;eau distillée chaude. La concentration finale de barbitone est 62.5mm. </li><li> Préparer une solution 3 en dissolvant 20.33gm de MgCl 2 et 4.41gm de CaCl2 dans 100 ml d&#39;eau distillée. La concentration finale de MgCl 2 et CaCl 2 est 2.18m et 440mm respectivement. </li><li> Mélanger les solutions 1 et 2 et laisser refroidir à température ambiante. </li><li> Une fois la solution combinée a refroidi, ajouter 1,25 ml de la solution 3 et ajuster le pH à 7.3 au 7.5 en utilisant HCl 1M. </li><li> Ajuster le volume final à 500 ml d&#39;eau distillée pour préparer une solution stock de 5x. </li><li> Pour préparer une solution 1x travail, diluer le stock de 01h05 à l&#39;eau distillée. </li></ol> Sensibilisation des hématies de mouton avec hémolysine <ol><li> Préparer l&#39;hémolysine tout d&#39;abord en le diluant avec VBS 01:50 </li><li> Pour ajouter 4ml de SRBC 6ml de VBS et mélanger délicatement par inversion </li><li> Centrifuger à 600g x 5 minutes </li><li> Jeter le surnageant et laver les cellules une autre 2 fois </li><li> Après le lavage final, centrifuger les cellules à 5 minutes x 900g pour emballer les cellules </li><li> Jeter le surnageant et remettre les cellules en quantité suffisante VBS pour préparer une solution de 10% soit 0,5 ml de cellules sont resuspendues dans emballée 5 ml de tampon </li><li> Goutte à goutte ajouter un volume égal d&#39;hémolysine (lapin anti-mouton d&#39;anticorps des globules rouges) pour les cellules tout en tourbillonnant en continu </li><li> Incuber à 30 ° C pendant 30 minutes dans un bain d&#39;eau </li><li> Mélanger délicatement les cellules toutes les 15 minutes </li><li> Sensibilisé SRBC peut être stockée pendant la nuit à 4 ° C </li></ol> CH 50 test <ol><li> Etiquette d&#39;une série de tubes en double avec 1:08, 1:16, 1:32, 1:64 et 1:128. </li><li> Préparer une série de deux dilutions successives de contrôle et de sérum à tester en VBS, en double exemplaire à chaque </li><li> Départ à 01h04 (100ml de sérum + 300ml VBS) et le transfert de 200ml d&#39;échantillon dans le tube suivant étiquetés. </li><li> Mélanger soigneusement entre les dilutions et 200ml transfert à la dilution suivante avec une pointe de pipette propre. </li><li> Répétez jusqu&#39;à ce que tous les cinq dilutions sont faites. </li><li> Jeter 200ml de la finale 1:128 dilution. </li><li> Ajouter 200ml de SRBC sensibilisés suspendus à tous les tubes. </li><li> Etiquette deux tubes séparés que de blanc et ajouter 200 ml d&#39;sensibilisés SRBC VBS + 200ml. Ces tubes mesurent lyse spontanée de la GRM en VBS. </li><li> Etiquette encore deux tubes séparés que lyse totale et ajouter 200 ml d&#39;sensibilisés SRBC + 200ml d&#39;eau distillée. </li><li> Mélanger délicatement tous les tubes. </li><li> Incuber à 37 ° C pendant 30 minutes dans un bain-marie le mélange après 15 minutes. </li><li> Centrifuger les échantillons à 1500 g pendant 5 minutes pour sédimenter les globules rouges. </li><li> Transfert de 100 ml de surnageant de chaque tube à un puits dans une plaque de 96 puits à fond plat. </li><li> Ajouter 100ml d&#39;eau distillée dans chaque puits. </li><li> Lire l&#39;absorbance des échantillons à 540nm en utilisant un spectrophotomètre assiette. </li></ol> Calculs <ol><li> Calculer l&#39;absorbance moyenne pour chaque échantillon </li><li> Soustraire l&#39;absorbance BLANC (lyse spontanée) de tous les échantillons </li><li> Calculez le% de lyse pour chaque dilution en utilisant la formule suivante: <img src="/files/ftp_upload/1923/1923eq1.jpg" alt="Équation" /></li><li> Tracer le pourcentage de lyse (axe vertical) en fonction de la dilution du sérum sur l&#39;axe horizontal. </li><li> Calculer la dilution nécessaire pour 50% d&#39;hémolyse pour le contrôle et le sérum à tester (figure 2). </li></ol> Les résultats représentatifs: La vidéo comprend un exemple de résultats représentatifs. En pratique, un sérum de contrôle est également géré dans le même temps que le sérum à tester et est traitée de la même manière. Ci-dessous (Tableau 1) sont des données de l&#39;échantillon à partir d&#39;un échantillon testé le sérum. Les données sont manipulées selon l&#39;équation présentée au paragraphe 5.3. <table border="1"><tbody><tr><td> Exemple </td><td> OD 540 </td><td> Moyenne </td><td></td><td></td></tr><tr><td> Vierge </td><td> 0.042, 0.044 </td><td> 0,043 </td><td></td><td></td></tr><tr><td> Lyse totale </td><td> 0.183, 0.183 </td><td> 0,183 </td><td></td><td></td></tr><tr><td colspan="5"></td></tr><tr><td> De dilution </td><td> OD 540 </td><td> Moyenne </td><td> Mean-Blank </td><td> % De lyse </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="Figure 1" /> Figure 1. L&#39;activation de la voie classique du complément. La voie classique est activée par la fixation des immunoglobulines M (IgM) ou des immunoglobulines G (IgG) sur la surface d&#39;une cellule cible. La portion Fc de l&#39;Ab se lie à C1q, C1r est activé, ce qui active à son tour une autre molécule de C1r qui, ensemble, d&#39;activer deux molécules de C1s. C1s clive désormais C4 qui expose le site de liaison pour C2, qui est aussi clivé. La liaison du C4b et C2a conduit à la formation d&#39;un complexe appelé C3-convertase. Ce complexe clive C3 formant désormais C3a et C3b, dont certains se combinent avec la C3 convertase de former une C5 convertase. Ce complexe agit maintenant sur C5, avec le C5b résultant liant à C6 ouverture de la formation du complexe d&#39;attaque membranaire (MAC). C5b6 agit sur C7, qui agissent en son tour sur le C8 et C9 en fin de compte aboutir à la formation de la finale de MAC. (Goldsby et al, 2003). <img src="http://www.jove.com/files/ftp_upload/1923/1923fig2.jpg" alt="Figure 2" /> Figure 2. Tracé des données d&#39;échantillon et le calcul du CH50 pour un contrôle et des échantillons de sérum à tester. (A), le pourcentage calculé (%) lyse de la fois le contrôle (•) et test () des échantillons de sérum sont tracées contre le facteur de dilution. (B) Pour le calcul de la lyse de 50%, une ligne est tracée à partir de la valeur pour cent à 50% jusqu&#39;à son intersection avec la ligne graphique, puis une ligne verticale est tiré vers le bas pour la dilution. Dans cet exemple, une dilution de 35 fois du contrôle et de 21,6 fois la dilution du sérum à tester sont nécessaires pour atteindre 50% de lyse. Ces données indiquent qu&#39;il ya moins de compléter présents dans le sérum à tester par rapport à la commande car il nécessitait moins de dilution avant la lyse de 50% a été atteint. L&#39;échantillon de contrôle montre également la lyse&gt; 100% à la dilution 1:8. Ceci est probablement dû à une lyse incomplète des SRBC par l&#39;eau distillée et plus efficace par la lyse des composants du complément.

<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>

Le CH 50 dosage est soumis à de nombreuses interférences. Certains SRBC sont plus fragiles que d&#39;autres, ce qui entraîne une hémolyse spontanée qui n&#39;est pas lié à compléter l&#39;activité. L&#39;affinité des anticorps de lapin varie d&#39;un lot à et d&#39;un fabricant à l&#39;autre, ce qui affecte la quantité d&#39;anticorps qui se lie à la GRM. En outre, le processus de sensibilisation SRBC avec des résultats d&#39;anticorps dans les cellules avec différentes quantités de revêteme...

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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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<tr>
<td>Serum</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Human serum</td>
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<tr>
<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>
<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

Le système du complément est un groupe de protéines qui lorsqu&#39;il est activé conduire à la lyse des cellules cibles et facilite la phagocytose par opsonisation. Composants du complément individuel peuvent être quantifiés mais cela ne donne aucune information quant à l&#39;activité de la voie. Le CH<sub&gt; 50</sub&gt; Est un test de dépistage pour l&#39;activation de la voie classique du complément (fig 1) et il est sensible à la réduction, l&#39;absence et / ou l&#39;inactivité de tout composant de la voie. Le CH<sub&gt; 50</sub&gt; Teste la capacité fonctionnelle des composants du complément sérique de la voie classique pour lyser des hématies de mouton (GRM) pré-enduites de lapin anti-mouton d&#39;anticorps des globules rouges (hémolysine). Lorsque recouvertes d&#39;anticorps SRBC sont incubées avec le sérum à tester, la voie classique du complément est activé et les résultats hémolyse. Si un composant du complément est absent, le CH<sub&gt; 50</sub&gt; Niveau sera égal à zéro, si un ou plusieurs composants de la voie classique sont diminués, le CH<sub&gt; 50</sub&gt; Sera diminué. Un volume fixe de façon optimale SRBC sensibilisés est ajouté à chaque dilution de sérum. Après incubation, le mélange est centrifugé et le degré d&#39;hémolyse est quantifié en mesurant l&#39;absorbance de l&#39;hémoglobine libérée dans le surnageant à 540nm. Le montant de l&#39;activité du complément est déterminé en examinant la capacité des différentes dilutions de sérum à tester afin de lyser SRBC revêtues d&#39;anticorps. Cette vidéo décrit les étapes impliquées dans l&#39;analyse expérimentale du niveau d&#39;activité complément de la voie classique du complément.

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

La voie classique est activée par l&#39;anticorps et culmine dans la lyse des cellules cibles. Le CH<sub&gt; 50</subTest&gt; fournit une mesure de l&#39;activité du complément d&#39;un échantillon de sérum. Cette vidéo montre les étapes impliquées dans la détermination de la CH<sub&gt; 50</sub&gt; D&#39;un échantillon de sérum, les calculs et l&#39;interprétation des résultats.

Mesurer le complément hémolytique 50% (CH 50) Activité de Sérum

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 vues.  University of South Australia. La voie classique est activée par l'anticorps et culmine dans la lyse des cellules cibles. Le CH 50 fournit une mesure de l'activité du complément d'un échantillon de sérum. Cette vidéo montre les étapes impliquées dans la détermination de la CH 50 D'un échantillon de sérum, les calculs et l'interprétation des résultats.

Vidéo: 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.

Appauvrissement des populations de cellules spécifiques par l&#39;épuisement Complément

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

Recherche

Immunologie et infection

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.

Détermination de la réactivité et le titre du sérum en utilisant un dosage d&#39;hémagglutination

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.

Mesure de l&#39;activité calpaïne dans des cellules fixées et de la Vie par cytométrie en flux

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.

Mesure de la charge bactérienne et les réponses immunitaires chez des souris infectées avec des 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.

Déterminer l&#39;activité phagocytaire des échantillons d&#39;anticorps cliniques

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.

La salive, les glandes salivaires, et Collection hémolymphe de tiques Ixodes scapularis

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.

Les méthodes de détection quantitative d&#39;anticorps induite par l&#39;activation du complément sur les globules rouges

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.

Mesure de l&#39;anaphylaxie locaux chez la souris

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.

Mesure de granulocytes et de monocytes phagocytose et oxydative Activité Burst dans le sang humain

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.

Dosages pour étudier le rôle de la vitronectine dans adhésion bactérienne et la résistance au sérum

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