Name: conjunctival commensal isolation and identification in mice
Uploaded: 2021-05-01T13:00:00
Description: Watch this Scientific Journal Video about Conjunctival Commensal Isolation and Identification in Mice at JoVE.com

I finanziamenti del P30 DK034854 hanno supportato VY, LB e studi nel Massachusetts Host-Microbiome Center e finanziamenti da NIH/NEI R01 EY022054 supportato MG.

Tutte le procedure che coinvolgono i topi seguono le linee guida del Comitato istituzionale per la cura e l'uso degli animali. Segui le linee guida sulla sicurezza di laboratorio (come indicato dal tuo dipartimento istituzionale per la salute e la sicurezza ambientale) quando lavori con microrganismi e materiali potenzialmente contaminati. Utilizzare recipienti di rifiuti appropriati e procedure di decontaminazione prima dello smaltimento di materiali contaminati potenzialmente a rischio biologico.
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<ol>
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E., Searcy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+mannitol+salt+agar+and+blood+agar+plates+for+identification+and+susceptibility+testing+of+Staphylococcus+aureus+in+specimens+from+cystic+fibrosis+patients.">Comparison of mannitol salt agar and blood agar plates for identification and susceptibility testing of Staphylococcus aureus in specimens from cystic fibrosis patients.</a> Journal of Clinical Microbiology. 44 (12), 4545-4546 (2006).</li><li>Siegman-Igra, Y., Azmon, Y., Schwartz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Milleri+group+streptococcus--a+stepchild+in+the+viridans+family.">Milleri group streptococcus--a stepchild in the viridans family.</a> European Journal of Clinical Microbiology and Infectious Diseases. 31 (9), 2453-2459 (2012).</li><li>Mohan, B., Zaman, K., Anand, N., Taneja, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aerococcus+Viridans:+A+Rare+Pathogen+Causing+Urinary+Tract+Infection.">Aerococcus Viridans: A Rare Pathogen Causing Urinary Tract Infection.</a> Journal of Clinical and Diagnostic Research. 11 (1), 1-3 (2017).</li><li>Senneby, E., Nilson, B., Petersson, A. 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J., 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=IL-1R+and+MyD88+contribute+to+the+absence+of+a+bacterial+microbiome+on+the+healthy+murine+cornea.">IL-1R and MyD88 contribute to the absence of a bacterial microbiome on the healthy murine cornea.</a> Frontiers in Microbiology. 9, 1117 (2018).</li><li>Ozkan, J., 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=Temporal+Stability+and+Composition+of+the+Ocular+Surface+Microbiome.">Temporal Stability and Composition of the Ocular Surface Microbiome.</a> Scientific Reports. 7 (1), 9880 (2017).</li><li>Oliver, J. 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J., 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=Culture-enriched+metagenomic+sequencing+enables+in-depth+profiling+of+the+cystic+fibrosis+lung+microbiota.">Culture-enriched metagenomic sequencing enables in-depth profiling of the cystic fibrosis lung microbiota.</a> Nature Microbiology. 5 (2), 379-390 (2020).</li><li>Raymond, F., 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=Culture-enriched+human+gut+microbiomes+reveal+core+and+accessory+resistance+genes.">Culture-enriched human gut microbiomes reveal core and accessory resistance genes.</a> Microbiome. 7, 56 (2019).</li><li>Peto, L., 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=Selective+culture+enrichment+and+sequencing+of+feces+to+enhance+detection+of+antimicrobial+resistance+genes+in+third-generation+cephalosporin+resistant+Enterobacteriaceae.">Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae.</a> PLoS One. 14 (11), 0222831 (2019).</li><li>Lauer, B. 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A causa dello stato paucibacterial della superficie oculare, molti laboratori hanno avuto difficoltà a isolare i commensaloculari 7,20, con conseguente basso numero di campioni con crescita, bassa abbondanza e bassa diversità8. Questo metodo migliora significativamente le pratiche di coltura standard4,21 con l'aggiunta di una fase di arricchimento, nonché un tampone per gli occh...

Lo scopo di questo protocollo è quello di migliorare l'isolamento specifico di microbi anaerobici aerobici e facultativi vitali e rari dalla congiuntiva oculare per caratterizzare il microbioma oculare. Studi approfonditi hanno profilato le comunità mucose commenali sulla pelle, l'intestino, le vie respiratorie e genitali e mostrano che queste comunità influenzano lo sviluppo del sistema immunitarioe la risposta 1,2,3. Le comunità commenali oculari hanno dimostrato di cambiare durante alcune patologie della malattia, come la malattia dell'occhiosecco 4,la sindrome di Sjogren5 e il diabete6. Tuttavia, la capacità di definire una tipica comunità commensale di superficie oculare è ostacolata dalla loro abbondanza relativamente bassa rispetto agli altri siti dimucosa 6,7,8. Ciò suscita una controversia sul fatto che esista un microbioma oculare residente e se esiste, se differisce dal microbioma cutaneo e, di conseguenza, il suo effetto locale sullo sviluppo e la risposta del sistema immunitario innato. Questo protocollo può aiutare a risolvere questa domanda.
Generalmente, gli approcci per definire la nicchia commensal oculare si basano sul sequenziamento e sulle tecnichebasate sulla cultura 4,7,9. 16 Il sequenziamento s rDNA e l'analisi BRISK7 mostrano una diversità più ampia rispetto alle tecniche basate sulla cultura, ma non sono in grado di distinguere tra microbi vivi e morti. Poiché la superficie oculare è ostile a molti microbi a causa delle proprietà antimicrobiche4 del film lacrimale che generano una vasta gamma di frammenti di DNA, gli approcci basati sul DNA rileveranno questi artefatti che possono distorcere i dati verso l'identificazione di batteri morti come commensal residenti piuttosto che contaminanti. Ciò si traduce in un'identificazione commensale aberrante e nella caratterizzazione dello spazio oculare come più elevato nell'abbondanza e nella diversità dei microbi10. Ciò rende difficile definire il microbioma oculare residente tramite metodi basati sul DNA. Considerando che le tecniche standard basate sulle impostazioni cultura non sono in grado di rilevare commensals perché il carico ètroppo basso 11. Il nostro metodo migliora le pratiche standard utilizzando un tampone sottile in grado di colpire la congiuntiva, evitando così la contaminazione dalla pelle vicina, così come il concetto che gli organismi vitali possono essere arricchiti da una breve coltura in mezzi densi di nutrienti con l'obiettivo di rianimare vitale ma non culturabile, oltre che arricchente per rari microbi vitali.
I risultati, l'abbondanza relativa di commensali oculari per tampone oculare, caratterizzano il microbioma residente congiuntiva e sono importanti per scopi comparativi. I nostri dati mostrano che c'è una differenza tra pelle e microbiota congiuntivale, così come una maggiore diversità con l'aumento dell'età e una differenza di abbondanza specifica per sesso. Inoltre, questo approccio ha trovato riproducibilmente differenze commenali nei topi knock-out12. Questo protocollo può essere applicato per descrivere il microbioma oculare che può variare a causa di pratiche di permanenza in gabbia, geografia o stato di malattia, così come gli effetti locali dei metaboliti commensali e dei prodotti sullo sviluppo e la risposta del sistema immunitario.

<table>
	<tbody>
		<tr>
			<td>0.1 to 10 &#181;l pipet tip</td>
			<td>USA Scientific</td>
			<td>1110-300</td>
			<td>autoclave before use</td>
		</tr>
		<tr>
			<td>0.5 to 10 &#181;l Eppendorf pipet</td>
			<td>Fisher Scientific</td>
			<td>13-690-026</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>Fisher Scientific</td>
			<td>BD309623</td>
			<td>1 syringe for each eye swab group</td>
		</tr>
		<tr>
			<td>1.5 ml Eppendorf tubes</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td>autoclave before use</td>
		</tr>
		<tr>
			<td>1000 &#181; ml pipet tip</td>
			<td>USA Scientific</td>
			<td>1111-2021</td>
			<td>autoclave before use</td>
		</tr>
		<tr>
			<td>200 to 1000&#181;l Gilson pipetman (P1000)</td>
			<td>Fisher Scientific</td>
			<td>F123602G</td>
			<td></td>
		</tr>
		<tr>
			<td>25 G needle</td>
			<td>Fisher Scientific</td>
			<td>14-826AA</td>
			<td>1 needle per eye swab group</td>
		</tr>
		<tr>
			<td>3 % Hydrogen Peroxide</td>
			<td>Fisher Scientific</td>
			<td>S25359</td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176; C Incubator</td>
			<td>Lab equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70 % Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>PX1840-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Ana-Sed Injection (Xylazine 100 mg/ml)</td>
			<td>Santa Cruz Animal Health</td>
			<td>SC-362949Rx</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BBL Gram Stain kit</td>
			<td>Fisher Scientific</td>
			<td>B12539</td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen Burner</td>
			<td>Lab equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clean paper towels</td>
			<td>Lab equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Batting/Sterile rolled cotton</td>
			<td>CVS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable 1 ml Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-711-9AM</td>
			<td>for Gram stain and catalase tests</td>
		</tr>
		<tr>
			<td>E.coli</td>
			<td>ATTC</td>
			<td>ATCC 8739</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-A3</td>
			<td>for Gram stain and catalase tests</td>
		</tr>
		<tr>
			<td>Ketamine (100mg/ml)</td>
			<td>Henry Schein</td>
			<td>9950001</td>
			<td></td>
		</tr>
		<tr>
			<td>Mac Conkey Agar Plates</td>
			<td>Fisher Scientific</td>
			<td>4321270</td>
			<td>store at 4 &#176;C until ready to use</td>
		</tr>
		<tr>
			<td>Mannitol Salt Agar</td>
			<td>Carolina Biological Supply</td>
			<td>784641</td>
			<td>Prepare plates according to mfr&#39;s instructions, store at 4 &#176;C for 1 week</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Jackson Labs</td>
			<td>C57/BL6J</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>08-757-12</td>
			<td>for Mannitol Salt agar plates</td>
		</tr>
		<tr>
			<td>RPI Brain Heart Infusion Media</td>
			<td>Fisher Scientific</td>
			<td>50-488525</td>
			<td>prepare according to directions and autoclave</td>
		</tr>
		<tr>
			<td>SteriFlip (0.22 &#181;m pore size polyester sulfone)</td>
			<td>EMD/Millipore, Fisher Scientifc</td>
			<td>SCGP00525</td>
			<td>to sterilize anesthesia</td>
		</tr>
		<tr>
			<td>Sterile Corning Centrifuge Tube</td>
			<td>Fisher Scientific</td>
			<td>430829</td>
			<td>anesthesia preparation</td>
		</tr>
		<tr>
			<td>Sterile mouse cage</td>
			<td>Lab equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tooth picks (round bamboo)</td>
			<td>Kitchen Essentials</td>
			<td></td>
			<td>autoclave before use and swab preparation</td>
		</tr>
		<tr>
			<td>Trypticase Soy Agar II with 5% Sheep&#39;s Blood Plates</td>
			<td>Fisher Scientific</td>
			<td>4321261</td>
			<td>store at 4 &#176;C until ready to use</td>
		</tr>
		<tr>
			<td>Vitek target slide</td>
			<td>BioMerieux Inc. Durham,NC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vitek-MS</td>
			<td>BioMerieux Inc. Durham,NC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vitek-MS CHCA matrix solution</td>
			<td>BioMerieux Inc. Durham, NC</td>
			<td>411071</td>
			<td></td>
		</tr>
		<tr>
			<td>Single use eye drops</td>
			<td>CVS Pharmacy</td>
			<td></td>
			<td>Bausch and Lomb Soothe Lubricant Eye Drops, 28 vials, 0.02 fl oz. each</td>
		</tr>
	</tbody>
</table>

conjunctival commensal isolation and identification in mice

La superficie oculare era un tempo considerata immune privilegiata e abiotica, ma recentemente sembra che ci sia una piccola ma persistente presenza commerciale. L'identificazione e il monitoraggio delle specie batteriche nella mucosa oculare sono stati impegnativi a causa della loro bassa abbondanza e della limitata disponibilità di una metodologia appropriata per la crescita e l'identificazione commensal. Esistono due approcci standard: metodi basati sulla cultura o sequenziamento del DNA. Il primo metodo è problematico a causa dei batteri recuperabili limitati e il secondo approccio identifica sia i batteri vivi che morti che portano a una rappresentazione aberrante dello spazio oculare. Abbiamo sviluppato un metodo robusto e sensibile per l'isolamento batterico basandoci su tecniche di coltivazione microbiologiche standard. Questa è una tecnica a base di tampone, utilizzando un tampone sottile "in-lab" realizzato che si rivolge alla congiuntiva inferiore, seguita da una fase di amplificazione per i generi anaerobici aerobici e facultativi. Questo protocollo ci ha permesso di isolare e identificare specie congiuntivali come Corynebacterium spp., Coagulase Negative Staphylococcus spp., Streptococcus spp. L'approccio è adatto a definire la diversità commenale nei topi in diverse condizioni di malattia.

I risultati rappresentativi di una piastra per tamponi per gli occhi che dimostra diversi metodi di placcatura sono raffigurati nella figura 3A che mostra isolati morfologicamente diversi dal mouse C57BL/6. Per ogni isolato distinto, le colonie sono state contate nella striscia e l'abbondanza relativa, unità di formazione delle colonie uniche (CTU) per tampone oculare, calcolate e tracciate a scopo di confronto. Per la caratterizzazione microbiologica, i batteri sono stati prelevati da sing...

Watch this Scientific Journal Video about Conjunctival Commensal Isolation and Identification in Mice at JoVE.com

Conjunctival Commensal Isolation and Identification in Mice

Qui è presentato un protocollo per l'isolamento e l'amplificazione di batteri commenali congiuntivali anaerobici aerobici e facultativi del topo utilizzando un tampone oculare unico e una fase di arricchimento basata sulla coltura con successiva identificazione con metodi microbiologici basati e spettrometria di massa MALDI-TOF.

Isolamento e identificazione congiuntivale dei commensali nei topi

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. ...

Questo metodo identifica microrganismi congiuntivali oculari vitali, il che è impegnativo a causa dell'ambiente ostile degli occhi. Può aiutare a rispondere se esiste un microbioma oculare e come la presenza batterica cambia nella malattia degli occhi. A differenza del sequenziamento del DNA, che identifica microrganismi sia vivi che non vivi, questo metodo identifica solo microrganismi vivi, risultando in una comprensione più chiara della comunità commensal oculare.Gli studi suggeriscono che le malattie oculari difficili da diagnosticare come l'occhio secco non autoimmune e autoimmune hanno una presenza batterica oculare unica. Questo metodo potrebbe essere utilizzato per diagnosticare le malattie degli occhi con firme microbiche distintive. Inizia con l'autoclave della quantità appropriata di batting di cotone e stuzzicadenti per il numero di topi da tamponare, quindi pizzica un pezzo di cotone lungo 1/2 centimetro e stuzzicandolo tirando fuori i bordi per formare un singolo strato poroso piatto che si ferma poco prima che la battuta cada a pezzi.Ruotare la battuta intorno a una delle estremità affilate dello stuzzicadenti tenendo leggermente il pezzo teso sulla punta dello stuzzicadenti mentre è attorcigliato. Il tampone per gli occhi finito avrà uno strato molto sottile di cotone allungato sulla punta che si estende a circa 1/2 a un centimetro di distanza dalla punta. Inserire i tamponi in un piccolo becher, tamponare lateralmente verso il basso, quindi coprirli e autoclavarli.Pulire l'area di lavoro con un disinfettante per ridurre al minimo la contaminazione e aliquota di 0,5 millilitri di infusione sterile di cuore cerebrale o mezzi BHI in tubi di micro centrifuga sterile da 1,5 millilitri etichettati. Fissare i tubi in un rack e mettere il rack sul ghiaccio. Impostare il flusso di lavoro da sinistra a destra a partire dalla stazione di anestetizzazione del mouse, che contiene la gabbia con i topi sperimentali, gabbia sterile vuota, anestesia a temperatura ambiente, ago calibro 25 e una siringa da un millilitro.Successivamente, preparare una stazione di tamponamento oculare che contenga il BHI aliquoto su ghiaccio, tamponi sterilizzati, contagocce lubrificanti, contenitore a rischio biologico, asciugamani di carta puliti e spray isopropanolo al 70%. Infine, impostare la stazione di placcatura con piastre di agar del sangue a temperatura ambiente, una pipetta da 10 microliter, punte monouso sterili da 10 microliter e un contenitore di rifiuti a rischio biologico. Assicurarsi che il mouse sia anestetizzato correttamente spremendo un pedante posteriore.Procedere solo se non c'è movimento. Assegnare una mano per gestire i topi anestetizzati e le altre mani per gestire il tampone oculare e la coltura. Rimuovere il mouse dalla gabbia e posizionarlo sopra la superficie di lavoro posizionata su un lato con l'occhio sinistro esposto.Spruzzare le mani guantate con isopropanolo e asciugarle con un tovagliolo di carta. Scoltare il tubo di micro centrifuga BHI etichettato con la mano di movimentazione dei supporti dedicata e posizionare i due indietro nel rack. Immergere la punta rivestita di cotone del tampone per gli occhi nel BHI, quindi ritirare il tampone dal tubo mentre ruotare la punta due volte contro la camera d'aria per rimuovere il liquido in eccesso e rimuoverlo.Con la mano di movimentazione del mouse, tenere delicatamente il mouse per la tracolla del collo. Con l'altra mano, posizionare la punta del tampone oculare contro la regione congiuntivale mediale dell'occhio sinistro. Deprimere leggermente il bulbo oculare e spostare il tampone in un movimento di lavaggio della finestra tra la palpebra inferiore e l'occhio 10 volte, mantenendo una pressione costante.Senza toccare la pelliccia, rimuovere delicatamente la punta del tampone perpendicolare al punto in cui è stata inserita. Posizionare il lato in cotone tampone verso il basso, direttamente in un tubo di micro centrifuga etichettato con supporti BHI. Applicare un colliro sull'occhio tamponato.Se lo si desidera, acquisire tamponi di pelle o pelliccia per campioni di controllo, sterilizzando i guanti in modo appropriato tra ogni tampone. Al termine, riportare il mouse nella gabbia. Lasciare riposare il tampone per 10-15 minuti sul ghiaccio, quindi sterilizzare le mani guantate e rimuovere il tampone mescolando la punta nel supporto per 10 rotazioni.Ritirare il tampone ruotando la punta contro la parete interna del tubo per cinque rotazioni e smaltirlo in un contenitore a rischio biologico. Ripetere il processo per ogni mouse. Arricchire il campione incubando il tubo staticamente per un'ora a 37 gradi Celsius.Durante l'incubazione, etichettare una piastra TSA a temperatura ambiente per tampone per occhio del mouse o tampone di controllo e dividerla a metà. Rimuovere i campioni arricchiti dall'incubatrice e metterli sul ghiaccio. Vortice brevemente i campioni da mescolare, quindi aliquota 10 microlitri del campione sulla piastra TSA e inclinare la piastra per formare una striscia.Ripeti questo due volte. Dall'altro lato della linea di demarcazione delle piastre, creare 10 punti con 10 microlitri di campione ciascuno. Incubare i piatti a 37 gradi Celsius per 18 ore, due giorni e quattro giorni in una camera pulita che impedisce alle piastre di agar di asciugarsi.Conta le colonie nelle strisce. Si noti morfologia e si calcolano le unità di formatura delle colonie per tampone per isolati morfologicamente simili. Guarda i punti in cui gli organismi unici non vengono catturati nelle strisce.Per facilitare la visualizzazione, abbiamo placcato i batteri densamente nelle placche centrali e giuste. In genere, abbiamo osservato molto meno batteri nelle placche del tampone oculare. Si noti che a volte, i tamponi oculari potrebbero non produrre batteri recuperabili.Qui viene mostrata una piastra rappresentativa per tamponi oculari con isolati morfologicamente diversi da un topo C57 nero a sei. Per ogni isolato distinto, le colonie furono contate nella striscia e l'abbondanza relativa fu calcolata e tracciata. Per la caratterizzazione microbiologica, i batteri sono stati prelevati da singole piastre per tamponi per topi per produrre una piastra TSA principale.Quando è apparsa la crescita, sono stati eseguiti ulteriori test per caratterizzare o identificare i microbi. La piastra principale è stata utilizzata per fornire abbastanza inoculo per espandere i rispettivi isolati. Per identificare gli isolati, una piastra TSA è stata striata e incubata durante la notte e è stata eseguita la MALDI-TOF MS.Esempi di isolati qui mostrati sono stati identificati come Streptococcus acidominimus e Aerococcus viridans. Livelli significativamente diversi di organismi commensali sono stati recuperati da maschi e femmine C57 neri sei topi. Streptococcus acidominimus, Aerococcus viridans coagula stafilococchi negativi ed E.coli sono stati isolati dai topi C57 neri sei N.Quando si tenta questo protocollo, tenere presente che il miglior risultato sarà ottenuto rivestendo il tampone sottilmente e prendendo tempo durante la fase del tampone oculare. Se l'accesso alla MALDI-TOF MS è limitato, gli isolati possono essere identificati da test microbiologici e biochimici.

conjunctival-commensal-isolation-and-identification-in-mice

Research

JoVE Journal

Immunology and Infection

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

Isolamento e analisi di Brain-sequestrati da leucociti<em&gt; Plasmodium berghei</em&gt; ANKA topi infettati

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Ricerca

Immunologia e infezioni

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

Rapida identificazione dei batteri Gram-negativi dal sangue brodo di coltura Uso MALDI-TOF

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

Isolamento, identificazione e purificazione di cellule murine timica epiteliali

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrofili Isolamento e analisi per determinare il loro ruolo nel linfoma a cellule sensibilità agli agenti terapeutici

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Identificazione di sequenze di localizzazione Plasmodesmal in proteine In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

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. ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Identificazione e isolamento di Oligopotent e dei progenitori mieloidi Lineage-commesso dal midollo osseo del topo

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.

Isolamento e identificazione di cellule immunitarie extravascolare del cuore

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. 
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.

Un modello di amministrazione orale Galleria mellonella a risposte immunitarie Innate Study Commensal-Induced

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is responsible for altering infection for many of these viruses. Thus, characterizing the nature of virus-bacteria binding is a foundational step needed for elucidating the mechanism(s) by which bacteria alter viral infection. For human norovirus, commensal bacteria enhance B cell infection. The virus directly binds to these bacteria, indicating that this direct interaction is involved in the mechanism of infection enhancement. A variety of techniques can be used to quantify interactions between bacteria and viruses including scintillation counting of radiolabeled viruses and polymerase chain reaction (PCR). Both methods require the use of live virus, which may need to be generated in the laboratory. Currently, none of the established in vitro culture systems available for human norovirus are robust enough to allow for generation of highly concentrated viral stocks. In lieu of live virus, virus-like particles (VLPs) have been used to characterize the interactions between norovirus and bacteria. Herein a flow cytometry method is described with uses virus specific antibodies to quantify VLP binding to gram-negative and gram-positive bacteria. Inclusion of both bacteria only and isotype controls allowed for optimization of the assay to reduce background antibody binding and accurate quantification of VLP attachment to the bacteria tested. High VLP:bacterium ratios result in VLPs binding to large percentages of the bacterial population. However, when VLP quantities are decreased, the percent of bacteria bound also decreases. Ultimately, this method can be employed in future experiments elucidating the specific conditions and structural components that regulate norovirus:bacterial interactions.

The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the population.