Supporto parziale da sovvenzioni a MRS è di scienze naturali e ingegneria Research Council del Canada (NSERC), l&#39;Harry Botterell Foundation for Neuroscience Research, ARC, e la ricerca Garfield Kelly cardiovascolari e Fondo di sviluppo. SRA è un destinatario del Premio Regina di ricerca dell&#39;Università e del Robert J. Wilson Fellowship. PJ è un destinatario del premio di Laurea della Regina e il Robert J. Wilson Fellowship. AG e SA sono i destinatari del premio di Laurea della Regina. SF è stato il destinatario della Borsa di Studio Ontario Laurea in Scienza e Tecnologia (OGSST). AG è il destinatario della Borsa di studio della Regina Franklin Bracken Graduate. SA è il destinatario della Borsa di studio della Regina RS McLaughlin Graduate.

1. Resuscitare i macrofagi congelati <ol><li> Prima di risorgere cellule congelate dal freezer -80 ° C, si ha la necessità di preparare un terreno di coltura sterile filtrata utilizzando Dulbecco Modified Eagle Medium (DMEM) supplementato con 10% siero fetale bovino (FBS) e 5 mg / mL di plasmocin. Plasmocin è una soluzione di antibiotico utilizzato nella nostra ricerca per eliminare e prevenire la contaminazione da micoplasma di colture cellulari. </li><li> Per un flaconcino di cellule congelate, si aggiunge 4 mL, 1 mL del mezzo di coltura per un CM 25 2 pallone di coltura cellulare in una cappa sterile contenimento rischio biologico. </li><li> Quando la fiala di cellule congelate sono prese dal congelatore ° -80 C, vengono scongelati rapidamente dal riscaldamento mano nella cappa sterile, che dura circa 2-3 minuti. </li><li> Non appena le cellule vengono scongelati, vengono rapidamente trasferiti nel pallone contenente il mezzo condizionato usando una pipetta sterile 1 ml. </li><li> Il pallone contenente le cellule si trovano in un tessuto culturale incubatore a 37 ° C e 5% di CO 2. </li><li> Le cellule controllato per la crescita e l&#39;adesione su base giornaliera fino a diventare il 75% al ​​90% confluenti utilizzando un microscopio invertito. </li><li> Durante l&#39;incubazione di colture cellulari, 12 vetrini millimetri circolare vengono sterilizzati con copertura in etanolo al 70% in un grande piatto di vetro petri per 1 ora nel quadro di contenimento rischio biologico, l&#39;alcol è poi rimosso e conservato per reusage, e le pareti di vetro bagnato sono lasciati esposti alla sterile filtrata flusso d&#39;aria nel mobile sotto un UV per 24 ore o fino a quando sono asciutti. </li></ol> 2. Le cellule placcatura per il saggio sialidasi <ol><li> Prima di placcatura le cellule, una singola scivolo sterile di vetro circolare viene accuratamente posizionato in un pozzetto di un Falcon 24-BD bene a fondo piatto con coperchio tessuto-cultura lastra di polistirene trattate utilizzando un sterilizzati curva fiammeggiante pieno, 4 &quot;, seghettata, pinza in acciaio inox . Per un pallone di cellule al 75% confluenza, di solito utilizzare 12 pozzetti della piastra di coltura. </li><li> Per le cellule macrofagi aderenti, il medium DMEM condizionato viene rimosso dal pallone con una pipetta da 5 ml sterile nel quadro di contenimento sterile. Lavare le cellule aderenti, una volta con 1x sterile salina Tris tamponata pH 7,4 a rimuovere qualsiasi supporto contenente siero, e aggiungere 1 ml di calcio-media liberi (CMF) tampone fosfato a pH 7.4 sufficiente a coprire le cellule. Le cellule si trovano nel 37 ° C incubatore per circa 10 minuti o fino a quando le cellule iniziano a sollevare nel pallone. Il pallone può essere cullati o agitato al fine di allentare le rimanenti cellule aderenti. </li><li> Per il 1 ml di cellule sospese nel pallone, aggiungere 3 ml di medium DMEM condizionata. Prendere 0,5 mL di cellule sospese e sterilmente loro trasferite alla piastra di coltura di tessuto contenente le 12 lastre di vetro circolare mm. Circa, 0,5 x 10 6 cellule vengono trasferite alle diapositive di vetro circolare. </li><li> La piastra di coltura delle cellule dei tessuti contenenti vengono incubate a 37 ° C in 5% di CO 2 per 24 ore al fine di consentire alle cellule di aderire alle diapositive di vetro circolare. </li></ol> 3. Sialidasi Assay 1. Rendere il controllo <ol><li> Posizionare un vetrino da microscopio (smerigliato: 1 fine, 1slide, 25 x 75 x 1 mm) nel quadro di contenimento o banco di laboratorio ed aggiungere mescolando in ordine sequenziale i seguenti composti: 1 ml DAKO + 1 ml di 4-MUNANA e infine + 3 ml di soluzione salina Tris tamponata pH 7.4 (TBS). </li><li> Dopo aver rimosso i mezzi di comunicazione da un pozzetto contenente le cellule, sollevare delicatamente il vetrino circolare di vetro con il &quot;4 pinza verso l&#39;angolo del pozzo. Rimuovere con cura il vetrino con la pinza e inserire il vetrino con le cellule si affaccia sulla soluzione miscela di il vetrino da microscopio. </li><li> Immediatamente, prendere lo scorrimento microcope ad un epi-microscopia a fluorescenza che è stato impostato per il filtro UV e ha una fotocamera digitale per catturare le immagini fluorescenti. </li><li> Messa a fuoco il microscopio in contrasto di fase fino a vedere le cellule e quindi passare alla modalità fluorescente. Scatta foto delle cellule sotto fluorescenti a 1 min, 2 min e 3 min. Prendete una singola immagine delle cellule in contrasto di fase. </li></ol> 2. Rendere il test positivo <ol><li> Spot i seguenti composti in ordine sequenziale su un altro vetrino da microscopio: 1 ml DAKO + 1 ml di 4-MUNANA + 2 ml di LPS + 1 ml di TBS. </li><li> Ripetere i passi da 3.1.2 a 3.1.4 di cui sopra. </li></ol> 3. Rendere il test positivo con Tamiflu inibitore della neuraminidasi <ol><li> Spot i seguenti composti nel seguente ordine sequenziale su un altro vetrino da microscopio: 1ml DAKO + 1ml di 4-MUNANA + 2 ml di LPS + 1 ml di Tamiflu. </li><li> Concentrazioni di Tamiflu sono pre-preparati in 1x salina Tris tamponata pH 7.4; la concentrazione finale dei composti in contatto con le cellule in diretta su vetrino circolare avrà un fattore di diluizione su 5. </li><li> Ripetere i passi da 3.1.2 a 3.1.4 di cui sopra. </li></ol> 4. Determinazione della concentrazione di inibitore necessaria per inibire il 50% delle attività sialidasi (IC 50) <ol><li> Per quantificare il fluorescenti che circonda le cellule, le immagini vengono analizzati utilizzando ImageJ 1.38x con l&#39;analizzare i plugin per la misurazione RBG. Prendere 50 letture casuali della fluorescenza che circonda le cellule e calcolare la media della fluorescenza totale. </li><li> L&#39;IC 50 è generato da un complotto della concentrazione di inibitore convertito in unità log (ad esempio, log ng / mL) su l&#39;asse x contro la fluorescenza media sul l&#39;asse y utilizzando la regressione lineare. </li></ol> 5. I segreti del successo <ol><li> Assicurarsi che le cellule in cultura sono esenti da contaminazione da micoplasma. Noi abitualmente uso Plasmocin in un terreno di coltura per il controllo per questo. </li><li> Il substrato artificiale neuraminidasi, 4-MUNANA, devono essere utilizzati preparati al momento. Il substrato preparato può essere utilizzato entro una settimana per il test sialidasi. </li><li> Se la linea cellulare ha una bassa espressione dei recettori sulla superficie cellulare, si può usare una dose più alta del ligando per la stimolazione. </li><li> Abbiamo anche sperimentato che le cellule diversi passaggi per più di 6 volte potrebbero perdere il loro fenotipo recettore. Fresco, le cellule devono essere coltivate risorto. </li></ol> 6. Rappresentante Risultati Vedi protocollo d&#39;animazione del saggio sialidasi con risultati rappresentante nel allegato <a href="http://www.jove.com/files/ftp_upload/2142/szewczuk_JoVE_sialidase_assay.pptx">file di Powerpoint</a> .

<ol>
	<li>Amith, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neu1+desialylation+of+sialyl+alpha-2,3-linked+beta-galactosyl+residues+of+TOLL-like+receptor+4+is+essential+for+receptor+activation+and+cellular+signaling.">Neu1 desialylation of sialyl alpha-2,3-linked beta-galactosyl residues of TOLL-like receptor 4 is essential for receptor activation and cellular signaling.</a> Cell Signal. 22, 314-324 (2010).</li><li>Amith, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependence+of+pathogen+molecule-induced+Toll-like+receptor+activation+and+cell+function+on+Neu1+sialidase.">Dependence of pathogen molecule-induced Toll-like receptor activation and cell function on Neu1 sialidase.</a> Glycoconjugate Journal. 26, 1197-1212 (2009).</li><li>Woronowicz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependence+of+neurotrophic+factor+activation+of+Trk+tyrosine+kinase+receptors+on+cellular+sialidase.">Dependence of neurotrophic factor activation of Trk tyrosine kinase receptors on cellular sialidase.</a> Glycobiology. 17, 10-24 (2007).</li><li>Jayanth, P., Amith, S. R., Gee, K., Szewczuk, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neu1+Sialidase+and+Matrix+Metalloproteinase-9+Cross-talk+is+Essential+for+Neurotrophin+Activation+of+Trk+Receptors+and+Cellular+Signaling.">Neu1 Sialidase and Matrix Metalloproteinase-9 Cross-talk is Essential for Neurotrophin Activation of Trk Receptors and Cellular Signaling.</a> Cell. Signal. 22, 1193-1205 (2010).</li><li>Finlay, T. M., Jayanth, P., Amith, S. R., Glimour, A., Guzzo, C., Gee, K., Beyaert, R., Szewczuk, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymoquinone+from+nutraceutical+black+cumin+oil+activates+Neu4+sialidase+in+live+macrophage,+dendritic,+and+normal+and+type+I+sialidosis+human+fibroblast+cells+via+GPCR+G&#945;i+proteins+and+matrix+metalloproteinase-9.">Thymoquinone from nutraceutical black cumin oil activates Neu4 sialidase in live macrophage, dendritic, and normal and type I sialidosis human fibroblast cells via GPCR G&#945;i proteins and matrix metalloproteinase-9.</a> Glycoconj J. 27, 329-348 (2010).</li><li>Seyrantepe, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Phagocytosis+in+Macrophages+by+Neuraminidase+1.">Regulation of Phagocytosis in Macrophages by Neuraminidase 1.</a> Journal of Biological Chemistry. 285, 206-215 (2010).</li></ol>

SRA e PJ contribuito in maniera uguale come primi autori.

Utilizzando il test di nuova concezione per rilevare l&#39;attività sialidasi in cellule vive macrofagi 2, abbiamo utilizzato questa tecnologia per rilevare l&#39;attività sialidasi in ligando indotta attività sialidasi in live BMC-2 cellule macrofagi in modo dose-dipendente e in live-2.4 DC cellule dendritiche , HEK-TLR4/MD2, HEK293, le cellule di adenocarcinoma mammario SP1, WT umana e 1140F01 e WG0544 tipo I fibroblasti sialidosi. Gli inibitori della neuraminidasi come il Tamiflu (oseltamivir fosfato) h...

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<td>DMEM</td>
<td>&nbsp;</td>
<td>Gibco, Rockville, MD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>4-MUNANA</td>
<td>&nbsp;</td>
<td>Biosynth International Inc., Itasca, IL, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fetal calf serum</td>
<td>&nbsp;</td>
<td>HyClone, Logan, Utah, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DakoCytomation, Fluorescent Mounting Media</td>
<td>&nbsp;</td>
<td>DAKO North America, Inc., Carpintenia, CA</td>
<td>S3023</td>
<td>15 mL</td>
</tr>
<tr>
<td>Tamiflu (Oseltamivir Phosphate)</td>
<td>&nbsp;</td>
<td>Hoffman La Roche</td>
<td>&nbsp;</td>
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detection of  neu1 sialidase activity in regulating toll-like receptor activation

Mammiferi recettori Toll-like (TLR) sono una famiglia di recettori che riconoscono pattern molecolari patogeni associati. Non solo i sensori di TLR cruciale microbica (ad esempio, virus, batteri e parassiti) infezioni, hanno anche un ruolo importante nella fisiopatologia delle malattie infettive, malattie infiammatorie, e forse anche nelle malattie autoimmuni. Così, l&#39;intensità e la durata delle risposte TLR contro le malattie infettive devono essere strettamente controllati. Ne consegue che la comprensione l&#39;integrità strutturale dei recettori del sensore, le loro interazioni ligando e componenti segnalazione è essenziale per la successiva protezione immunologica. Sarebbe inoltre fornire importanti opportunità per la modifica delle malattie attraverso la manipolazione del sensore. Anche se le vie di segnalazione di sensori TLR sono ben caratterizzati, i parametri che controllano le interazioni tra i sensori e loro ligandi sono ancora poco definiti. Abbiamo recentemente identificato un nuovo meccanismo di attivazione TLR dal suo ligando naturale, che non è stato precedentemente osservato 1,2. Suggerisce che ligando indotta attivazione TLR è strettamente controllato dalla NEU1 attivazione sialidasi. Abbiamo anche riferito che NEU1 regola strettamente recettori neurotrofina come TrkA e TrkB 3, che coinvolgono NEU1 e la matrice metalloproteinasi-9 (MMP-9) cross-talk in complesso con i recettori 4. Il dosaggio sialidasi è stato inizialmente utilizzato per trovare un romanzo ligando, Timochinone, l&#39;attivazione di Neu4 sialidasi sulla superficie cellulare di macrofagi, cellule dendritiche e le cellule di fibroblasti via GPCR proteine ​​Gαi e MMP-9 5. Per i recettori TLR, i nostri dati indicano che NEU1 sialidasi è già in complessi con i recettori TLR-2, -3 e -4, ed è indotta su di legame a uno dei recettori. Attivato NEU1 idrolizza sialidasi sialyl α-2 ,3-legati residui distante da ligando β-galattosiltransferasi vincolante per rimuovere impedimento sterico di TLR-4 dimerizzazione, MyD88/TLR4 reclutamento complesso, l&#39;attivazione NFkB e pro-infiammatorie risposte delle cellule. In un rapporto collaborativo, sialidasi NEU1 è stato dimostrato che regolano la fagocitosi in macrofagi 6. Nel loro insieme, il saggio sialidasi ci ha fornito spunti potente per i meccanismi molecolari del ligando indotta da attivazione del recettore. Sebbene l&#39;esatta relazione tra NEU1 sialidasi e l&#39;attivazione di TLR, recettori Trk deve ancora essere completamente chiarito, che rappresenterebbe un nuovo approccio pionieristico o per percorsi di regolazione cellulare.

Watch this Scientific Journal Video about Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation at JoVE.com

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Il test sialidasi è un approccio semplice tecnica che chiarire romanzo meccanismo molecolare (s) di sensori TLR di infezioni microbiche e di coinvolgimento nelle malattie infiammatorie a livello dei recettori sulla superficie cellulare di macrofagi dal vivo.

Rilevazione di attività NEU1 sialidasi nella regolamentazione Toll-like l&#39;attivazione del recettore

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

detection-neu1-sialidase-activity-regulating-toll-like-receptor

Research

JoVE Journal

Immunology and Infection

11.5K Views.  Queen's University - Kingston, Ontario. Il test sialidasi è un approccio semplice tecnica che chiarire romanzo meccanismo molecolare (s) di sensori TLR di infezioni microbiche e di coinvolgimento nelle malattie infiammatorie a livello dei recettori sulla superficie cellulare di macrofagi dal vivo.

Video: Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Combinazione di nastro adesivo a base di campionamento e di fluorescenza In situ</emIbridazione> per il rilevamento rapido di Salmonella Su prodotti freschi

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Ricerca

Immunologia e infezioni

Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like allergic reactions 1. In clinical practice, diagnosis of drug hypersensitivity is mainly performed by patient history, as skin testing is not reliable and oral provocation testing bears life-threatening risks for the patient 2. Hence, evidence for an underlying IgE-mediated pathomechanism is hard to obtain. 
Here, we present an in vitro method based on the use of human basophils derived from drug-hypersensitive patients that mimics the allergic effector reaction in vivo. As basophils of drug-allergic patients carry IgE molecules specific for the culprit drug, they become activated upon IgE receptor crosslinking and release allergic effector molecules. The activation of basophils can be monitored by the determination of the upregulation of CD63 surface expression using flow cytometry 3. 
In the case of low molecular weight drugs, conjugates are designed to enable IgE receptor crosslinking on basophils. As depicted in Figure 1, two representatives of NSAIDs, PP and DF, are covalently bound to human serum albumin (HSA) via a carboxyl group reacting with the primary amino group of lysine residues. DF carries an intrinsic carboxyl group and, thus, can be used directly 4, whereas a carboxyl group-containing derivative of PP had to be organochemically synthesized prior to the study 1.
The coupling degree of the low molecular weight compounds on the protein carrier molecule and their spatial distribution is important to guarantee crosslinking of two IgE receptor molecules. The here described protocol applies high performance-size exclusion chromatography (HPSEC) equipped with a sequential refractive index (RI) and ultra violet (UV) detection system for determination of the coupling degree.
As the described methodology may be applied for other drugs, the basophil activation test (BAT) bears the potential to be used for the determination of IgE-mediated mechanisms in drug hypersensitivity. Here, we determine PP hypersensitivity as IgE-mediated and DF hypersensitivity as non-IgE-mediated by BAT.

Basophil activation test is a potent tool for the detection of IgE-dependent allergies in vitro. Here, an optimized protocol for basophil activation test is used to investigate drug hypersensitivity. A method for the efficient production of covalent drug-protein conjugates and their physicochemical characterization is described.

L&#39;attivazione dei basofili di prova per le Indagini di IgE-mediata Meccanismi di ipersensibilità a farmaci

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like ...

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4 and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7. 
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-&kappa;B, which initiates transcription of numerous genes that are critical for immune responses8. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-&kappa;B pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9.

We describe use of ImageStream technology (<a href="http://www.amnis.com/" target="_blank">www.amnis.com</a>), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

Imaging quantitativa di Lineage specifici Toll-like receptor-mediate Signaling in monociti e cellule dendritiche da piccoli campioni di sangue umano

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

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 ...

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&amp;#946; in Human Monocyte-derived Dendritic Cells

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

Attivazione e misurazione dei NLRP3 inflammasome all&#39;attività utilizzando IL-1β in cellule dendritiche umane derivate da monociti

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Isolamento di murini peritoneale macrofagi di effettuare analisi di espressione genica Al Toll-like recettori stimolazione

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

L&#39;isolamento e l&#39;attivazione di linfociti murini

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

Flusso analisi di citometria di Natural Killer cellulare Lytic attività nel Sangue umano intero

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.