Vorremmo riconoscere Mathias S. Dick, Roland F. Dreier e Sebastian Rühl per la discussione. Questo lavoro è stato supportato da un FNS Professorship PP00P3_139120 / 1 e Università di Basilea concessione progetto ID2153162 a PB

1. Digitonin Assay con mCherry esprimono Salmonella (FACS-based Analysis) <ol><li> Batteri Preparazione Nota: Salmonella wild-type SL1344 (streptomicina resistente) che esprime mCherry da un plasmide (pFPV codifica mCherry sotto il promotore RPSM) viene utilizzato in questo test. Fagocitosi batterica e la proliferazione in cui non alterata dalla espressione costitutiva di mCherry (dati non riportati) 16. <ol><li> Inoculare il ceppo 1 giorno prima della infezione in 3 ml di terreno LB addizionato con streptomicina (90 mg / ml) e ampicillina (100 ug / ml, mCherry esprimendo vettore) in un agitatore a 37 ° CO / N. </li></ol></li><li> Placcatura cellule per l&#39;infezione. <ol><li> Seed wild-type BMDMs in un piatto ben 24 con una densità di 1,25 x 10 5 cellule / pozzetto in 1 ml di terreno macrofagi primaria (DMEM supplementato con 10% di siero fetale bovino (FCS, inattivato al calore, 56 ° C, 30 min ), 1% HEPES, 1% di aminoacidi non essenziali (NEAA), 1% di L-glutammina e il 10% L929 surnatante (medium condizionato da macrofagi fattore stimolante le colonie (M-CSF) che producono cellule L929). Pozzi seme secondo la tabella 1 per tenere conto di condizioni individuali. Aggiungere coprioggetto ai pozzetti utilizzati come controlli (Tabella 1). </li><li> Incubare le cellule durante la notte in un incubatore a 37 ° C e 5% CO 2. </li></ol></li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td></td><td></td><td colspan="2"> Permeabilization </td><td colspan="3"> Colorazione </td></tr><tr><td></td><td> Vetrino di vetro </td><td> Salmonella </td><td> Digitonin </td><td> Saponina </td><td> anti-Salmonella </td><td> anti-calnexina </td><td> anti-PDI </td></tr><tr><td> A1 (campione) </td><td> - / + * </td><td> + </td><td> + </td><td></td><td> + </td><td></td><td></td></tr><tr><td> A2 (campione) </td><td> - / + * </td><td> + </td&gt; <td> + </td><td></td><td> + </td><td></td><td></td></tr><tr><td> A3 (campione) </td><td> - / + * </td><td> + </td><td> + </td><td></td><td> + </td><td></td><td></td></tr><tr><td> A4 (nessun controllo permeabilizzazione) </td><td> - / + * </td><td> + </td><td></td><td></td><td> + </td><td></td><td></td></tr><tr><td> A5 (controllo completo permeabilizzazione) </td><td> - / + * </td><td> + </td><td></td><td> + </td><td> + </td><td></td><td></td></tr><tr><td> B1 (colorate per calnexina) </td><td> + </td><td></td><td></td><td></td><td></td><td> + </td><td></td></tr><tr><td> B2 (colorate per calnexina) </td><td> + </td><td></td><td> + </td><td></td><td></td><td> + </td><td></td></tr><tr><td> B3 (colorate per calnexina) </td><td> + </td><td></td><td></td><td> + </td><td></td><td> + </td><td></td></tr><tr><td> C1 (macchiatoper PDI) </td><td> + </td><td></td><td></td><td></td><td></td><td></td><td> + </td></tr><tr><td> C2 (colorate per PDI) </td><td> + </td><td></td><td> + </td><td></td><td></td><td></td><td> + </td></tr><tr><td> C3 (colorate per PDI) </td><td> + </td><td></td><td></td><td> + </td><td></td><td></td><td> + </td></tr></tbody></table> Tabella 1: schema di placcatura per midollo osseo macrofagi derivati ​​(BMDMs) in un formato a 24 pozzetti per la successiva infezione da Salmonella, permeabilizzazione, e la colorazione * A seconda se protocollo 1 o il protocollo 2 è fatto.. <ol start="3"><li> Preparare i batteri per l&#39;infezione. <ol><li> Il giorno successivo, determinare la concentrazione di Salmonella nella cultura overnight diluendo cultura 1:10 in PBS e misurando la OD 600 contro un unico controllo PBS. Questo assicura che la OD 600 è misurata nella lgamma inear dello spettrofotometro. </li><li> Calcolare la concentrazione di Salmonella per ml. Un OD600 di 1 corrisponde a 1 x 10 9 batteri. </li><li> Diluire i batteri in media macrofagi per raggiungere una concentrazione di Salmonella 1,25 x 10 6 cellule / ml. </li></ol></li><li> L&#39;infezione di cellule con Salmonella. <ol><li> Rimuovere media da BMDMs. Aggiungere 1 ml di terreno macrofagi di pozzetti di controllo non infetti (B1-B3, C1-C3). Aggiungere 1 ml di sospensione Salmonella nei pozzetti A1 - A5 per raggiungere una molteplicità di infezione (moi) di 10 batteri per cellula. </li><li> Centrifugare la piastra per 15 min a 300 xg a 37 ° C per sincronizzare l&#39;infezione. Trasferire la lastra in un incubatore a 37 ° C e 5% CO 2. </li></ol></li><li> Uccidere Salmonella extracellulare con Gentamicina. <ol><li> A 1 ora dopo l&#39;infezione, rimuovere la piastra da incubatore e aggiungere 0,1 ml di terreno macrofagi contenenti 0,1 mg / ml Gentamicinaper uccidere i batteri extracellulari. Aggiungere gentamicina a tutti i pozzetti, anche ai controlli non infetti, per garantire che tutte le cellule sono trattate nello stesso modo. Trasferire la lastra in un incubatore a 37 ° C e 5% CO 2. </li></ol></li><li> Lavare le cellule. <ol><li> A 2 ore dopo l&#39;infezione, rimuovere piatto da incubatore e lavare tutti i pozzetti 2x con 1 ml di DMEM pianura (pre-riscaldato a 37 ° C) e sostituirlo con media macrofagi (pre-riscaldato a 37 ° C), contenente 10 mg / ml &#160; Gentamycin per prevenire la crescita di tutte le rimanenti batteri extracellulari. Trasferire la lastra in un incubatore a 37 ° C e 5% CO 2. </li></ol></li><li> Preparazione tamponi freschi con detergenti e anticorpi. <ol><li> Poco prima del punto di tempo desiderato di analisi, preparare una soluzione madre fresco digitonina a 1 mg / ml in KHM Buffer (acetato di potassio 110 mM, HEPES 20 mM, MgCl 2 2 mM, pH 7,3). Filtrare il brodo e diluire in KHM tampone a una concentrazione di lavorodi 50 mg / ml di digitonina. Inoltre, preparare una soluzione di 0,1% in saponina KHM Buffer, anti-anticorpo Salmonella accoppiato al FITC (CSA-1-FITC) a 1/500, anti-calnexin a 1/100 o anti-PDI a 1/100 in KHM Buffer integrato con 3% BSA. </li></ol></li><li> Permeabilizzazione cellulare. <ol><li> Rimuovere la piastra da incubatore e lavare i pozzetti 3x con 0,5 ml di KHM Buffer (pre-riscaldato a 37 ° C). Rimuovere KHM Buffer e aggiungere 0,25 ml pianura KHM tampone ai pozzetti A4 e B1 / C1, KHM tampone con digitonina nei pozzetti A1 - A3 e B2 / C2 o KHM tampone con saponina di pozzi A5 e B3 / C3 (secondo la tabella 1). Incubare per esattamente 1 minuti a temperatura ambiente e immediatamente lavare tutti i pozzetti 3x con 0,5 ml di KHM Buffer (pre-riscaldato a 37 ° C). </li></ol></li><li> Colorazione anticorpo primario. <ol><li> Rimuovere il tampone KHM e aggiungere le soluzioni di anticorpo primario (capra anti- Salmonella -FITC, 250 microlitri / bene, 0.1 mg / ml) per i pozzetti (Figura 1 </strong&gt;, Tabella 1). Incubare la piastra per 15 minuti in un incubatore a 37 ° C e 5% CO 2. </li><li> Lavare le cellule 1x con 1 ml di PBS. </li></ol></li><li> Cellule infettate (pozzi A1 - A5): analisi FACS <ol><li> Lavare le cellule 3x con PBS e aggiungere 0,5 ml di ghiacciata PBS contenente 0,1% Triton. Trasferire in una provetta 5 ml FACS con tappo colino cella per rompere aggregati di cellule. I campioni in ghiaccio. </li><li> Analizzare i campioni di un FACS. Utilizzando i controlli completamente permeabilizzate, impostare la porta per i batteri totali basati su avanti e scatter laterale (FSC / SSC) in primo luogo, e il segnale mCherry (610 nm ± 20 nm filtro). </li><li> Avanti, analizzare il segnale FITC nella popolazione batterica totale utilizzando i controlli permeabilizzate completamente permeabilizzate e non per impostare le porte per i batteri citosoliche (FITC +, mCherry +) e batteri vacuolari (FITC, mCherry +) (Figura 3). </li><li> Con impostare le porte, analizzare i campioni e determinare la percentuale di rispetto citosolicobatteri vacuolari utilizzando il software FlowJo secondo il protocollo del produttore. </li></ol></li><li> Controlli permeabilizzazione non infette (pozzi B1 - B3, C1 - C3): microscopia <ol><li> Fissazione <ol><li> Rimuovere PBS e aggiungere 250 ml di PFA 4% in PBS. Incubare per 10 minuti a 37 ° C. </li><li> Lavare i pozzetti 2x con 1 ml di PBS e aggiungere 250 ml di 0,1 M glicina in PBS per 10 minuti per placare il fissativo. Lavare i pozzetti 2x con 1 ml di PBS. </li></ol></li><li> Colorazione anticorpo secondario. <ol><li> Controlli permeabilizzazione colorare per 1 ora a RT con appropriati anticorpi secondari accoppiati a fluorofori in PBS integrato con 0,1% saponina e 3% BSA a cellule completamente permeabilize. </li><li> Lavare coprioggetto 3x con 1 ml di PBS e montare le lamelle per analisi a mezzo di montaggio con DAPI (1,5 mg / ml). </li></ol></li><li> Analisi Microscopia. <ol><li> Analizzare i coprioggetti con i controlli a 63X di ingrandimento 40X o utilizzando un fluo confocaleMicroscopio rescence. Se la permeabilizzazione ha funzionato, non ci dovrebbe essere alcuna colorazione per le cellule non-permeabilizzate, calnexina colorazione per entrambe le celle digitonin- e saponina-permeabilizzate e PDI colorazione solo per le celle saponina permeabilizzate (Figura 4). </li></ol></li></ol></li></ol> 2. Digitonin Assay con Unlabeled Salmonella (Analisi basata Microscopia-) <ol><li> Preparazione batteri per O / N culture. <ol><li> Seminare senza etichetta Salmonella wild-type SL1344 (streptomicina resistente) 1 giorno prima della infezione in 3 ml di terreno LB addizionato con streptomicina (90 mg / ml) in un agitatore a 37 ° CO / N. </li><li> Placcatura cellule per l&#39;infezione. <ol><li> BMDMs seme in un piatto ben 24 contenente vetrini sterili ad una densità di 1,25 x 10 5 cellule / pozzetto in 1 ml di terreno macrofagi (DMEM integrato con il 10% di siero fetale bovino (FCS, de-integrato, 56 ° C,30 min), 1% HEPES, 1% acidi L-non-essenziale amino (NEAA), 1% L-glutammina e il 10% L929 surnatante). Pozzi seme secondo la tabella 1 per tenere conto di condizioni individuali. </li><li> Incubare le cellule O / N in un incubatore a 37 ° C e 5% CO 2. </li></ol></li><li> Preparare i batteri per l&#39;infezione. <ol><li> Il giorno dopo, a determinare la concentrazione di Salmonella nella / cultura O N diluendo la cultura 1:10 in PBS e misurando l&#39;OD 600 nei confronti di un solo controllo PBS. Questo assicura che la OD 600 è misurato nel range lineare dello spettrofotometro. </li><li> Determinare la concentrazione di Salmonella per millilitro. Un OD 600 di 1 corrisponde a 1 x 10 9 batteri. </li><li> Diluire i batteri in media macrofagi per raggiungere una concentrazione di Salmonella 1,25 x 10 6 cellule / ml. </li></ol></li><li> L&#39;infezione di cellule con Salmonella. <ol&gt; <li> Rimuovere media da tutti i pozzetti. Aggiungere 1 ml di terreno macrofagi pianura di pozzetti di controllo non infetti (B1 - B3, C1 - C3). Aggiungere 1 ml di sospensione Salmonella nei pozzetti A1 - A5 per raggiungere una molteplicità di infezione (moi) di 10 batteri per cellula. </li><li> Centrifugare la piastra per 15 min a 300 xg a 37 ° C per sincronizzare l&#39;infezione. Trasferire la lastra in un incubatore a 37 ° C e 5% CO 2. </li></ol></li><li> Uccidere Salmonella extracellulare con Gentamicina. <ol><li> A 1 ora dopo l&#39;infezione, rimuovere la piastra da incubatore e aggiungere 0,1 ml di terreno macrofagi contenenti 1 mg / ml di gentamicina per uccidere i batteri extracellulari tutti i pozzetti. Trasferire la lastra in un incubatore a 37 ° C e 5% CO 2. </li></ol></li><li> Lavare le cellule. <ol><li> A 2 ore dopo l&#39;infezione, rimuovere la piastra da incubatore e lavare i pozzetti 3x con 1 ml di mezzo fresco macrofagi contenente 10 mg / ml Gentamycin evitarela crescita di tutte le rimanenti batteri extracellulari. Trasferire la lastra in un incubatore a 37 ° C e 5% CO 2. </li></ol></li><li> Preparazione tamponi freschi con detergenti e anticorpi. <ol><li> Poco prima del punto di tempo desiderato di analisi, preparare una soluzione madre fresco digitonina a 1 mg / ml in KHM Buffer (acetato di potassio 110 mM, HEPES 20 mM, MgCl 2 2 mM, pH 7,3). Filtrare il brodo e diluire in KHM tampone a una concentrazione di lavoro di 50 mg / ml di digitonina. </li><li> Inoltre, preparare una soluzione di 0,1% in saponina KHM Buffer, anti- Salmonella -FITC (CSA-1, 0,1 mg / ml), anti-calnexin a 1/100 o anti-PDI a 1/100 in KHM Buffer integrato con 3% di BSA (vedi tabella materiali per la concentrazione di anticorpi). </li></ol></li><li> Permeabilizzazione cellulare. <ol><li> Rimuovere la piastra da incubatore e lavare i pozzetti 3x con 0,5 ml di KHM Buffer (pre-riscaldato a 37 ° C). Rimuovere KHM Buffer e aggiungere 0,25 ml pianura KHM Bufferper pozzi A4 e B1 / C1, KHM Buffer con digitonina nei pozzetti A1 - A3 e B2 / C2 o KHM tampone con saponina di pozzi A5 e B3 / C3 (secondo la tabella 1). Incubare per esattamente 1 minuti a temperatura ambiente e immediatamente lavare tutti i pozzetti 3x con 0,5 ml di KHM Buffer (pre-riscaldato a 37 ° C). </li></ol></li><li> Colorazione anticorpo primario. <ol><li> Rimuovere il tampone KHM e aggiungere le soluzioni di anticorpo primario (capra anti- Salmonella CSA1-FITC, 250 microlitri / bene, 0.1 mg / ml) per i pozzetti (Figura 2, Tabella 1). Incubare la piastra per 15 minuti in un incubatore a 37 ° C e 5% CO 2. Lavare 3x con 1 ml di PBS. </li></ol></li><li> Fissazione. <ol><li> Rimuovere PBS e aggiungere 250 ml di PFA 4% in PBS. Incubare per 10 minuti a 37 ° C. </li><li> Lavare i pozzetti 2x con 1 ml di PBS e aggiungere 250 ml di 0,1 M glicina in PBS per 10 minuti per placare il fissativo. Lavare i pozzetti 2x con 1 ml di PBS. </li></ol></li><li> TotaleSalmonella colorazione dei campioni infetti. <ol><li> Lavare 3x coprioggetto con PBS con 0,1% saponina / 3% BSA e incubare i campioni con un anticorpo anti-CSA1 (capra anti-CSA1, 0,1 mg / ml, topo anti-LPS funziona pure, 0,1 mg / ml) per 1 ora a RT in PBS con 0,1% saponina / 3% BSA. </li><li> Lavare 3x in PBS con 0,1% saponina / 3% BSA. Incubare i campioni con un anticorpo anti-capra secondario accoppiato al fluoroforo di scelta in PBS con 0,1% saponina / 3% BSA per 30 minuti a RT al buio. </li><li> Lavare coprioggetto 3x con 1 ml di PBS e montare le lamelle per analisi a mezzo di montaggio con DAPI (1,5 mg / ml). </li></ol></li><li> Analisi Microscopia. <ol><li> Acquisire dati con un microscopio confocale contando Salmonella citosolica (anti-Salmonella -FITC positivo) e totale Salmonella (doppia positivo) (Figura 5). Controlli permeabilizzazione dovrebbe mostrare: nessuna colorazione per le cellule non-permeabilizzate, calnexina scontenente per le cellule permeabilizzate con digitonina e PDI colorazione per le celle saponina permeabilizzate (Figura 4). Wells A4 e A5 serviranno controlli interni per la colorazione Salmonella. </li></ol></li></ol></li></ol>

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	<li>Kumar, Y., Valdivia, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leading+a+sheltered+life:+intracellular+pathogens+and+maintenance+of+vacuolar+compartments.">Leading a sheltered life: intracellular pathogens and maintenance of vacuolar compartments.</a> Cell Host Microbe. 5, 593-601 (2009).</li><li>Chong, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+early+phagosomal+stage+of+Francisella+tularensis+determines+optimal+phagosomal+escape+and+Francisella+pathogenicity+island+protein+expression.">The early phagosomal stage of Francisella tularensis determines optimal phagosomal escape and Francisella pathogenicity island protein expression.</a> Infect Immun. 76, 5488-5499 (2008).</li><li>Mellouk, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shigella+subverts+the+host+recycling+compartment+to+rupture+its+vacuole.">Shigella subverts the host recycling compartment to rupture its vacuole.</a> Cell Host Microbe. 16, 517-530 (2014).</li><li>Checroun, C., Wehrly, T. D., Fischer, E. R., Hayes, S. F., Celli, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy-mediated+reentry+of+Francisella+tularensis+into+the+endocytic+compartment+after+cytoplasmic+replication.">Autophagy-mediated reentry of Francisella tularensis into the endocytic compartment after cytoplasmic replication.</a> Proc Natl Acad Sci U S A. 103, 14578-14583 (2006).</li><li>Broz, P., Monack, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newly+described+pattern+recognition+receptors+team+up+against+intracellular+pathogens.">Newly described pattern recognition receptors team up against intracellular pathogens.</a> Nat Rev Immunol. 13, 551-565 (2013).</li><li>Hagar, J. A., Powell, D. A., Aachoui, Y., Ernst, R. K., Miao, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+LPS+activates+caspase-11:+implications+in+TLR4-independent+endotoxic+shock.">Cytoplasmic LPS activates caspase-11: implications in TLR4-independent endotoxic shock.</a> Science. 341, 1250-1253 (2013).</li><li>Ogawa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escape+of+intracellular+Shigella+from+autophagy.">Escape of intracellular Shigella from autophagy.</a> Science. 307, 727-731 (2005).</li><li>Haraga, A., Ohlson, M. B., Miller, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonellae+interplay+with+host+cells.">Salmonellae interplay with host cells.</a> Nat Rev Microbiol. 6, 53-66 (2008).</li><li>Birmingham, C. L., Smith, A. C., Bakowski, M. A., Yoshimori, T., Brumell, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+controls+Salmonella+infection+in+response+to+damage+to+the+Salmonella-containing+vacuole.">Autophagy controls Salmonella infection in response to damage to the Salmonella-containing vacuole.</a> J Biol Chem. 281, 11374-11383 (2006).</li><li>Malik-Kale, P., Winfree, S., Steele-Mortimer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bimodal+lifestyle+of+intracellular+Salmonella+in+epithelial+cells:+replication+in+the+cytosol+obscures+defects+in+vacuolar+replication.">The bimodal lifestyle of intracellular Salmonella in epithelial cells: replication in the cytosol obscures defects in vacuolar replication.</a> PLoS One. 7, e38732 (2012).</li><li>Zhao, Y. O., Khaminets, A., Hunn, J. P., Howard, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+the+Toxoplasma+gondii+parasitophorous+vacuole+by+IFNgamma-inducible+immunity-related+GTPases+(IRG+proteins)+triggers+necrotic+cell+death.">Disruption of the Toxoplasma gondii parasitophorous vacuole by IFNgamma-inducible immunity-related GTPases (IRG proteins) triggers necrotic cell death.</a> PLoS Pathog. 5, e1000288 (2009).</li><li>Meunier, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase-11+activation+requires+lysis+of+pathogen-containing+vacuoles+by+IFN-induced+GTPases.">Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-induced GTPases.</a> Nature. 509, 366-370 (2014).</li><li>Yamamoto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cluster+of+interferon-gamma-inducible+p65+GTPases+plays+a+critical+role+in+host+defense+against+Toxoplasma+gondii.">A cluster of interferon-gamma-inducible p65 GTPases plays a critical role in host defense against Toxoplasma gondii.</a> Immunity. 37, 302-313 (2012).</li><li>Degrandi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+guanylate+binding+protein+2+(mGBP2)+controls+Toxoplasma+gondii+replication.">Murine guanylate binding protein 2 (mGBP2) controls Toxoplasma gondii replication.</a> Proc Natl Acad Sci U S A. 110, 294-299 (2013).</li><li>Mariathasan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+activation+of+the+inflammasome+by+caspase-1+adaptors+ASC+and+Ipaf.">Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf.</a> Nature. 430, 213-218 (2004).</li><li>Knodler, L. A., Nair, V., Steele-Mortimer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+cytosolic+Salmonella+in+epithelial+cells.">Quantitative assessment of cytosolic Salmonella in epithelial cells.</a> PLoS One. 9, e84681 (2014).</li><li>Ng, H., Smith, D. J., Nagley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+flow+cytometry+to+determine+differential+redistribution+of+cytochrome+c+and+Smac/DIABLO+from+mitochondria+during+cell+death+signaling.">Application of flow cytometry to determine differential redistribution of cytochrome c and Smac/DIABLO from mitochondria during cell death signaling.</a> PLoS One. 7, e42298 (2012).</li><li>Krawczyk, E., Suprynowicz, F. A., Sudarshan, S. R., Schlegel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+orientation+of+the+human+papillomavirus+type+16+E5+oncoprotein.">Membrane orientation of the human papillomavirus type 16 E5 oncoprotein.</a> J Virol. 84, 1696-1703 (2010).</li><li>Keller, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+measurements+of+vacuolar+rupture+caused+by+intracellular+pathogens.">Single cell measurements of vacuolar rupture caused by intracellular pathogens.</a> J Vis Exp. , e50116 (2013).</li></ol>

We have nothing to disclose.

Permeabilizzazione differenziale è un metodo facile e robusto per analizzare e quantificare la distribuzione subcellulare di batteri patogeni tra compartimenti vacuolari e citoplasma. Lo stesso test è stato utilizzato con successo con batteri come Francisella novicida 2,4 e Shigella flexneri 12. Tuttavia, dal momento che molti patogeni intracellulari alterare o modificare le strutture endomembrane ospitanti, la robustezza della permeabilizzazione digitonina dovrà essere determi...

Batteri patogeni intracellulari replicano direttamente nel citoplasma della cellula ospite, o in scomparti vacuolari specializzati 1. Durante la fase iniziale dell&#39;infezione maggior patogeni vengono internalizzati mediante fagocitosi da cellule specializzate (come macrofagi) o promuovendo attivamente il proprio assorbimento nelle cellule non fagocitiche. Nelle cellule fagocitiche, phagosome normalmente fonde con lisosomi per formare un vano degradativa, dove vengono digeriti particelle fagocitati. Batteri citosolici specializzati, come ad esempio Francisella novicida o Shigella flexneri, fuga degrado phagosomal inducendo la rottura del phagosome e successivamente fuga nel 2,3 citoplasma. Ciò richiede meccanismo di virulenza associata come la Francisella patogenicità Island (FPI) o la Shigella flexneri T3SS, che inietta le proteine ​​effettrici che promuovono vacuolar rottura 2-4. Le caratteristiche citosol della cellula ospiteun numero di recettori pattern recognition conservati che normalmente riconoscono la presenza di agenti patogeni e inducono immunitaria innata segnalazione 5. Inoltre, xenophagy, una forma antimicrobica dell&#39;autofagia può degradare batteri che entrano nel citosol. Batteri citosoliche sono normalmente ben adattati per smussare o aggirare queste risposte utilizzando diverse strategie. Ad esempio, Francisella modifica le sue LPS per non farsi riconoscere host e Shigella impedisce l&#39;autofagia reclutamento utilizzando secrete proteine ​​effettrici 6,7. Un&#39;altra strategia per sfuggire degradazione lisosomiale è impiegato dal modello vacuolare patogeno Salmonella enterica sierotipo Typhimurium, di seguito la Salmonella typhimurium. Salmonella utilizza un T3SS per iniettare effettori che rimodellano phagosome iniziale in sua nicchia intracellulare, la Salmonella -contenenti vacuolo (SCV) 8,9. Manipolazione continua di percorsi di accoglienza ènecessaria per mantenere questo vacuolo attraverso l&#39;assunzione di lipidi e altre sostanze nutritive per il vacuolo. In effetti, un mutante Sifa di Salmonella non riesce a mantenere la stabilità vacuolar, ed entra nel citoplasma delle cellule ospiti poche ore dopo l&#39;infezione, che si traduce nell&#39;attivazione di percorsi immunitario innato e l&#39;autofagia reclutamento 8. Fuga vacuolare di Salmonella varia a seconda del tipo di cellula che gli studi e numerose relazioni hanno dimostrato che nelle cellule epiteliali anche WT Salmonella può sfuggire la SCV e hyperreplicate nel citosol 10. Recenti rapporti indicano che la diagnosi immunitario innato di patogeni vacuolari dipende anche dalla capacità di accoglienza di riconoscere e destabilizzare vacuoli da organismi patogeni che contiene (PCV) 11-14. Dato che sia l&#39;host o batteri genotipo possono influenzare la distribuzione dei batteri intracellulari tra vacuolare e compartimento citosolico, è necessario quantificare il numero di vacuolare vs.batteri citosolici. Poiché, in configurazioni più sperimentali cellule ospiti sono infettate con più di un batterio, e successivamente può ospitare diversi batteri in differenti compartimenti subcellulari, è necessaria una tecnica che consente la quantificazione del livello di singoli batteri. Permeabilizzazione differenziale (noto anche come test di protezione phagosomal) fornisce la presente risoluzione 2,4,10,12. Il saggio si basa sulla permeabilizzazione selettiva della membrana plasmatica della cellula ospite con la digitonina detersivo, che lascia membrane vacuolare intatta e consente così la colorazione selettiva di batteri citosolici con anticorpi. Qui forniamo due protocolli per la quantificazione dei citosolica e vacuolare Salmonella typhimurium con permeabilizzazione differenziale. Il principio di questo metodo è descritto nella Figura 1: di midollo osseo macrofagi derivati ​​(BMDMs) sono infettati con fase stazionaria mCherry esprimono Salmonella. Fase stazionaria Salmonella bisogno to essere utilizzato in quanto downregulate l&#39;espressione della SPI-1 T3SS, la cui attività sarebbe altrimenti essere riconosciuto dal inflammasome NLRC4 e indurre la morte delle cellule rapido (pyroptosis) del BMDMs 15. Dopo l&#39;infezione le cellule sono lavate e trattati con 50 ug / ml di digitonina per 1 minuto. Le cellule vengono nuovamente lavati immediatamente e incubate con anticorpi anti-Salmonella accoppiati a FITC marcare batteri con accesso al citosol. Dopo un ulteriore lavaggio cellule passo vengono lisate e la percentuale di batteri mCherry + (vacuolare) e FITC + / + mCherry (citosolica) è determinata da FACS. Riportiamo anche un adattamento di questo metodo che può essere utilizzato se non ceppi proteine ​​esprimono fluorescenti sono disponibili (Figura 2). I passaggi aggiuntivi vengono introdotte dopo l&#39;etichettatura FITC in cui le cellule sono fissi e completamente permeabilizzate. Successivamente tutti i batteri sono macchiati con anticorpi anti-Salmonella e anticorpi secondari corrispondenti. Detezione viene poi eseguita al microscopio invece di analisi FACS.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Digitonin</td>
			<td>Sigma</td>
			<td>D5628</td>
			<td>50ug/mL</td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>mpbio</td>
			<td>219998380</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Life Technologies</td>
			<td>15630</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Acetate</td>
			<td>Sigma</td>
			<td>791733</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Salmonella CSA-1-FITC</td>
			<td>KPL</td>
			<td>01-91-99-MG</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>anti-Salmonella CSA-1</td>
			<td>KPL</td>
			<td>02-91-99-MG</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>anti-Calnexin</td>
			<td>Enzo Lifesciences</td>
			<td>SPA-860D</td>
			<td>1/100</td>
		</tr>
		<tr>
			<td>anti-PDI</td>
			<td>Enzo Lifesciences</td>
			<td>SPA-890</td>
			<td>1/100</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma</td>
			<td>47036</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield mounting medium</td>
			<td>Vectorlabs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti Rabbit antibody-488</td>
			<td>Molecular Probes</td>
			<td>A-11070</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma</td>
			<td>G8898</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Life Technologies</td>
			<td>15710-49</td>
			<td>100ug/mL and 10ug/mL</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Promega</td>
			<td>H5141</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>20012-019</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D6429</td>
			<td></td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Amimed</td>
			<td>5-13K00-H</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM700 Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>imaging done at 63x</td>
		</tr>
		<tr>
			<td>FACS Fortessa</td>
			<td>BD technologies</td>
			<td></td>
			<td>detection mCherry 610 nm</td>
		</tr>
		<tr>
			<td>FACS Fortessa</td>
			<td>BD technologies</td>
			<td></td>
			<td>detection FITC 530 nm</td>
		</tr>
	</tbody>
</table>

quantification of cytosolic vs. vacuolar salmonella in primary macrophages by differential permeabilization

Batteri patogeni intracellulari possono replicarsi nel citosol o in vacuoli-patogeni contenenti specializzati (PCV). Per raggiungere il citosol, batteri come Shigella flexneri e Francisella novicida devono indurre la rottura del phagosome. Al contrario, Salmonella typhimurium replica in un vano vacuolare, conosciuto come Salmonella vacuole -contenenti (SCV). Tuttavia alcuni mutanti di Salmonella non riescono a mantenere l&#39;integrità SCV e sono quindi rilasciate nel citoplasma. La percentuale di citosolico contro batteri vacuolare a livello di singoli batteri può essere misurata permeabilizzazione differenziale, noto anche come test fagosoma protezione. L&#39;approccio fa uso della proprietà di detersivo digitonina di legarsi selettivamente colesterolo. Poiché la membrana plasmatica contiene più colesterolo che altre membrane cellulari, digitonina può essere utilizzato per permeabilize selettivamente la membrana plasmatica lasciando membrane intracellulariintatto. In breve, dopo l&#39;infezione con l&#39;agente patogeno che esprimono una proteina marcatore fluorescente (es mCherry tra gli altri), la membrana plasmatica delle cellule ospiti è permeabilizzate con una breve incubazione in digitonina contenente tampone. Le cellule vengono quindi lavate ed incubate con un anticorpo primario (accoppiato ad un fluoroforo di scelta) diretto contro il batterio di scelta (ad esempio anti-Salmonella -FITC) e nuovamente lavati. Se si utilizzano i batteri non marcati, un passaggio aggiuntivo può essere fatto, in cui tutte le membrane vengono permeabilizzate e tutti i batteri colorate con un anticorpo corrispondente. Dopo la colorazione, la percentuale di vacuolari e citosolici batteri può essere quantificata mediante FACS o microscopia contando eventi singoli o doppi-positive. Qui forniamo dettagli sperimentali per l&#39;uso di questa tecnica con il batterio Salmonella typhimurium. Il vantaggio di questo saggio è che, a differenza di altre analisi, fornisce una quantificazione del livello di singola bacteria, e se analizzata al microscopio fornisce il numero esatto di citosolico e batteri vacuolari in una data cellula.

Figura 1 e Figura 2 mostra gli schemi del test digitonina descritti nel protocollo 1 e 2 del protocollo che illustrano le fasi critiche del protocollo e dei risultati ottenuti. La figura 3 mostra tipici FACS risultati. I controlli positivi e negativi vengono utilizzati per impostare le porte per mCherry + / batteri FITC (vacuolar Salmonella) e mCherry + / FITC + batteri (citosolico Salmonella). Sulla base di queste porte la percentuale di batteri citosolici e vacuolari pu?...

Watch this Scientific Journal Video about Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization at JoVE.com

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

Quantificazione dei citosolico vs. vacuolare<em&gt; Salmonella</em&gt; In primarie macrofagi di Permeabilization differenziale

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

quantification-cytosolic-vs-vacuolar-salmonella-primary-macrophages

Research

JoVE Journal

Immunology and Infection

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

10.8K Views.  University of Basel. We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

Video: Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses1. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation2. This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency4. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively5. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.
Contraction of smooth muscle is generally regulated by the cytosolic Ca2+ concentration ([Ca2+]i) plus sensitivity to Ca2+, of the contractile elements in response to changes in the environment surrounding the cell6. [Ca2+]i is determined by the combination of the movement of Ca2+ through plasma membrane ligand or voltage gated Ca2+ channels and the release and uptake of Ca2+ from internal stores. Cytosolic Ca2+ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction8. However, the sensitivity of this pathway to Ca2+ can be regulated by the MLC phosphatase (MLCP)9. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.
Here, we present a method to evaluate changes in [Ca2+]i over time in isolated, perfused lymphatics in order to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca2+]i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca2+]i was determined by loading lymphatics with the ratiometric, Ca2+-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

We introduce an approach to evaluate the cytosolic Ca2+ concentration in isolated lymphatics to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction.

Misura della Ca citosolico 2 + In isolati Linfatici contrattile

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

Ricerca

Immunologia e infezioni

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Misure singole cellule di Rottura Vacuolar causate da patogeni intracellulari

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

High-throughput Assay per Fenotipo<em&gt; Salmonella enterica</em&gt; Typhimurium Associazione, Invasion, e replica nei macrofagi

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Trasfezione altamente efficiente di umani THP-1 da macrofagi Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

Proteomica Profiling dei macrofagi da 2D elettroforesi

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

Migrazione phagosome e velocità misurata in Live primarie macrofagi umani infettati con HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

Caratterizzazione fenotipica dei macrofagi dal rene di ratto mediante citometria di flusso

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolamento di Salmonella typhimurium-contenente fagosomi dai macrofagi

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Time-lapse Imaging 3D della fagocitosi dai macrofagi del topo

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought serious public safety concerns. Among the risk factors, antibody-dependent enhancement (ADE) poses the most significant threat, as the recent re-emergence of the Zika virus (ZIKV) is primarily in areas where the population has been exposed and is in a state of pre-immunity to other closely related flaviviruses, especially dengue virus (DENV). Here, we describe a protocol for quantifying the effect of human serum antibodies against DENV on ZIKV infection in primary human cells or cell lines.

We describe a method to evaluate the effect of pre-existing immunity against dengue virus on the Zika virus infection by using human serum, primary human cells, and infection quantification by quantitative real-time polymerase chain reaction.

Quantificazione di esaltazione anticorpo-dipendente del Virus in cellule umane primarie Zika

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought ...