L&#39;autore desidera ringraziare la signorina Lora Matthews per eseguire la tecnica e il signor Paolo Pastore per la cinematografia. Questo progetto è stato finanziato dalla University of South Australia, il programma di Medicina di Laboratorio.

Preparazione di 5x Veronal Buffered Saline (VBS) <ol><li> Per preparare il veronal Buffered Saline (VBS), tre soluzioni separate devono essere preparati. </li><li> 1 Preparare la soluzione sciogliendo 21.25gm di NaCl e 0.94gm Barbitone di sodio in 350 ml di acqua distillata. Le concentrazioni finali di NaCl e sodio Barbitone sono 1,02 e 13 mm rispettivamente. </li><li> Preparare la soluzione 2 sciogliendo 1.44gm di Barbitone in 125ml di acqua distillata calda. La concentrazione finale di Barbitone è 62,5 mm. </li><li> 3 Preparare la soluzione sciogliendo 20.33gm di MgCl 2 e 4.41gm di CaCl 2 in 100 ml di acqua distillata. La concentrazione finale di MgCl 2 e CaCl 2 è 2.18m e 440mm rispettivamente. </li><li> Mix di soluzioni 1 e 2 e raffreddare a temperatura ambiente. </li><li> Una volta che la soluzione combinata si è raffreddato, aggiungere 1.25ml di soluzione al 3 e regolare il pH a 7,3-7,5 usando HCl 1M. </li><li> Regolare il volume finale di 500ml con acqua distillata per preparare una soluzione 5x magazzino. </li><li> Per preparare una soluzione 1x di lavoro, diluire il magazzino 1:05 con acqua distillata. </li></ol> Sensibilizzazione dei globuli rossi di pecora con emolisina <ol><li> Preparare il emolisina da prima che diluendo 1:50 con VBS </li><li> Per 4ml di SRBC aggiungere 6 ml di VBS e mescolare delicatamente per inversione </li><li> Centrifugare a 600g x 5 minuti </li><li> Eliminare il supernatante e lavare le cellule altre 2 volte </li><li> Dopo l&#39;ultimo lavaggio, centrifugare le cellule a 900g x 5 minuti per confezionare le cellule </li><li> Eliminare il supernatante e risospendere le cellule in VBS sufficiente per preparare una soluzione al 10% cioè 0,5 ml di cellule confezionati sono risospese in 5 ml di tampone </li><li> Goccia a goccia aggiungere un volume equivalente di emolisina (coniglio anti-pecora rossa anticorpi di globuli rossi) per le cellule, mentre vorticose continua </li><li> Incubare a 30 ° C per 30 minuti in un bagno d&#39;acqua </li><li> Mescolare delicatamente le cellule ogni 15 minuti </li><li> Sensibilizzate SRBC possono essere memorizzati notte a 4 ° C </li></ol> CH 50 test <ol><li> Etichetta una serie di tubi in duplice copia con 1:8, 1:16, 1:32, 1:64 e 1:128. </li><li> Preparare una serie di due diluizioni seriali di controllo e di siero in esame in ogni VBS in duplice copia </li><li> Inizia 1:4 (100 ml di siero + 300 ml VBS) e il trasferimento da 200 ml di campione nella provetta etichettata successiva. </li><li> Mescolare accuratamente tra le diluizioni e 200ml trasferimento alla diluizione successiva con un puntale fresca. </li><li> Ripetere fino a quando tutti e cinque diluizioni sono fatti. </li><li> Eliminare 200ml dalla finale 1:128 diluizione. </li><li> Aggiungere 200 ml di sospensione SRBC sensibilizzati a tutti i tubi. </li><li> Etichetta due tubi separati come BLANK e aggiungere 200 ml di sensibilizzati SRBC + 200 ml VBS. Questi tubi misurerà lisi spontanea del SRBC in VBS. </li><li> Etichetta altri due tubi separati come lisi TOTALE e aggiungere 200 ml di sensibilizzati SRBC + 200 ml di acqua distillata. </li><li> Mescolare delicatamente tutti i tubi. </li><li> Incubare a 37 ° C per 30 minuti a bagnomaria mescolando dopo 15 minuti. </li><li> Centrifugare i campioni a 1.500 g per 5 minuti per sedimentare i globuli rossi. </li><li> Trasferimento 100ml di surnatante da ciascuna provetta per un pozzo in un piatto fondo ben 96 piatti. </li><li> Aggiungere 100 ml di acqua distillata in ciascun pozzetto. </li><li> Leggere l&#39;assorbanza dei campioni a 540nm utilizzando uno spettrofotometro piatto. </li></ol> Calcoli <ol><li> Calcolare l&#39;assorbanza media per ogni campione </li><li> Sottrarre l&#39;assorbanza del bianco (lisi spontanea) da tutti i campioni </li><li> Calcolare la lisi% per ogni diluizione utilizzando la seguente formula: <img src="/files/ftp_upload/1923/1923eq1.jpg" alt="Equazione" /></li><li> Tracciare la lisi percentuale (asse verticale) contro la diluizione del siero sull&#39;asse orizzontale. </li><li> Calcolare la diluizione necessaria per il 50% emolisi per il controllo e siero (Fig. 2). </li></ol> Rappresentante dei risultati: Il video contiene un esempio di risultati rappresentativi. In pratica, un siero di controllo viene eseguito anche allo stesso tempo come il siero in esame e viene trattato nella stessa maniera. Qui di seguito (Tabella 1) è un dato campione da un campione testato siero. I dati sono manipolati secondo l&#39;equazione presentata in 5.3. <table border="1"><tbody><tr><td> Campione </td><td> OD 540 </td><td> Dire </td><td></td><td></td></tr><tr><td> Vuoto </td><td> 0,042, 0,044 </td><td> 0,043 </td><td></td><td></td></tr><tr><td> Lisi 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> Diluizione </td><td> OD 540 </td><td> Dire </td><td> Significa-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="Figura 1" /> Figura 1. Attivazione della via classica del complemento. La via classica è attivata dal legame con immunoglobuline M (IgM) o immunoglobuline G (IgG) sulla superficie della cellula bersaglio. La porzione Fc della Ab lega al C1q, C1r è attivata e questo a sua volta attiva un&#39;altra molecola di C1r che, insieme, attivare due molecole di C1s. C1s si unirà ora C4 che espone il sito di legame per la C2, che è anche spaccati. Il legame di C4b e C2a porta alla formazione di un complesso chiamato C3 convertasi. Questo complesso si unirà ora C3 formando C3a e C3b, alcune delle quali si combinano con la C3 convertasi formando una C5 convertasi. Questo complesso agisce ora in C5, con la conseguente C5b legame con C6 di avviare la formazione del complesso di attacco alla membrana (MAC). C5b6 agisce sul C7, che a loro volta agiscono su C8 e C9 in ultima analisi, causando la formazione della finale MAC. (Goldsby et al, 2003). <img src="http://www.jove.com/files/ftp_upload/1923/1923fig2.jpg" alt="Figura 2" /> Figura 2. Rappresentazione grafica dei dati di esempio e il calcolo del CH50 per un controllo e campione di siero. (A), la percentuale calcolata (%) lisi sia del controllo (•) e test () campioni di siero sono tracciati contro il fattore di diluizione. (B) Per calcolare la lisi del 50%, viene disegnata una linea dal valore percentuale del 50% fino ad intersecare con le linee del grafico e poi una linea verticale viene disegnata fino alla diluizione. In questo esempio, è 35 volte diluizione del controllo e il 21,6 volte diluizione del siero in esame sono tenuti a raggiungere il 50% lisi. Questi dati indicano che c&#39;è meno complemento presenti nel siero in esame rispetto al controllo, come ha richiesto meno di diluizione prima 50% lisi è stato raggiunto. Il campione di controllo mostra anche&gt; 100% lisi alla diluizione 1:8. Questo è molto probabilmente dovuto alla lisi incompleta del SRBC dall&#39;acqua distillata e più efficiente lisi dai componenti del complemento.

<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>

Il CH 50 saggio è soggetta a molte interferenze. Alcuni SRBC sono più fragili di altre, con conseguente emolisi spontanea che non è correlata a completare l&#39;attività. L&#39;affinità degli anticorpi di coniglio varia da lotto a lotto e da un costruttore all&#39;altro, questo influisce sulla quantità di anticorpo che si lega alla GRP. Inoltre, il processo di sensibilizzazione SRBC con risultati anticorpi nelle cellule con quantità diverse di rivestimento degli anticorpi del GRP. Raccolta dei campioni...

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<td>Sheep red blood cells</td>
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<td>Use at 1% final</td>
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<td>Serum</td>
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<td>Human serum</td>
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<td>Rabbit Anti-Sheep Haemolytic serum (RBCs), Unconjugated</td>
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<td>C12HSB</td>
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<td>96 well flat bottom plate</td>
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<td>Sarstedt</td>
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<td>Veronal buffered saline</td>
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measuring the 50% haemolytic complement (ch50) activity of serum

Il sistema del complemento è un gruppo di proteine ​​che, quando attivato portare a bersaglio la lisi cellulare e facilita la fagocitosi attraverso opsonisation. Componenti complementari individuali possono essere quantificati comunque questo non fornisce alcuna informazione circa l&#39;attività della via. Il CH<sub&gt; 50</sub&gt; È un test di screening per l&#39;attivazione della via classica del complemento (Fig. 1) ed è sensibile alla riduzione, assenza e / o di inattività di qualsiasi componente del percorso. Il CH<sub&gt; 50</sub&gt; Verifica la capacità funzionale dei componenti del complemento del siero della via classica per lisare i globuli rossi di pecora (GRP) pre-rivestiti con coniglio anti-pecora anticorpi dei globuli rossi (emolisina). Quando legate agli anticorpi SRBC vengono incubati con siero, la via classica del complemento si attiva ed i risultati emolisi. Se un componente complementare è assente, il CH<sub&gt; 50</subLivello&gt; sarà pari a zero, se uno o più componenti della via classica sono diminuiti, il CH<sub&gt; 50</sub&gt; Sarà diminuito. Un volume fisso di GRP sensibilizzati in modo ottimale è aggiunto ad ogni diluizione del siero. Dopo l&#39;incubazione, la miscela viene centrifugata e il grado di emolisi viene quantificato misurando l&#39;assorbanza della emoglobina rilasciato nel supernatante a 540nm. La quantità di attività complementare viene determinata esaminando la capacità delle varie diluizioni del siero per lisare SRBC rivestiti di anticorpi. Questo video illustra le fasi sperimentali coinvolti nell&#39;analizzare il livello di attività complemento della via classica del complemento.

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 via classica è attivata da anticorpi e culmina nella lisi delle cellule bersaglio. Il CH<sub&gt; 50</subTest&gt; fornisce una misura dell&#39;attività complemento di un campione di siero. Questo video dimostra i passi coinvolti nella determinazione del CH<sub&gt; 50</sub&gt; Di un campione di siero, i calcoli e l&#39;interpretazione dei risultati.

Misura del Complemento 50% emolitica (CH 50) Attività di siero

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 Views.  University of South Australia. La via classica è attivata da anticorpi e culmina nella lisi delle cellule bersaglio. Il CH 50 fornisce una misura dell'attività complemento di un campione di siero. Questo video dimostra i passi coinvolti nella determinazione del CH 50 Di un campione di siero, i calcoli e l'interpretazione dei risultati.

Video: 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.

L&#39;esaurimento delle popolazioni di cellule specifiche da deplezione del complemento

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

Ricerca

Immunologia e infezioni

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.

Determinazione della reattività e Titolo di siero usando un test dell&#39;emoagglutinazione

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.

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

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Measuring 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.

Misurazione della carica batterica e Risposte immunitarie in topi infettati con 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.

Determinazione dell&#39;attività fagocitaria dei Campioni anticorpi clinici

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.

Saliva, le ghiandole salivari, e Collection emolinfa dalle zecche 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.

Metodi per quantitativa individuazione di anticorpi indotta da attivazione del complemento sui globuli rossi

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.

Misurare Anafilassi locali nei topi

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.

Misurazione dei granulociti e dei monociti fagocitosi e ossidativo attività Burst nel sangue umano

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.

Saggi per studiare il ruolo di Vitronectin nell'adesione batterica e sulla resistenza del siero

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