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

Finansiering fra P30 DK034854 støttede VY, LB og undersøgelser i Massachusetts Host-Microbiome Center og finansiering fra NIH/NEI R01 EY022054 støttede MG.

Alle procedurer, der involverer mus, følger retningslinjerne fra Udvalget for Institutionel Dyrepleje og -brug. Følg retningslinjerne for laboratoriesikkerhed (som anvist af din institutionelle miljøsundheds- og sikkerhedsafdeling), når du arbejder med mikroorganismer og potentielt forurenede materialer. Brug passende affaldsbeholdere og dekontamineringsprocedurer forud for bortskaffelse af potentielt biofarlige forurenede materialer.
1. Forberedelse af øjenprøve, opsætning af arbejdsomr?...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory Experiments in Microbiology. , (2010).</li><li>Reiner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalase+Test+Protocol.">Catalase Test Protocol.</a> American Society for Microbiology. , (2010).</li><li>UK SMI. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Standards for Microbiology Investigation. UK SMI. , (2014).</li><li>Sharp, S. 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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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxic+effect+of+calcium+alginate+swabs+on+Neisseria+gonorrhoeae.">Toxic effect of calcium alginate swabs on Neisseria gonorrhoeae.</a> Journal of Clinical Microbiology. 26 (1), 54-56 (1988).</li><li>Dubois, 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=Performances+of+the+Vitek+MS+matrix-assisted+laser+desorption+ionization-time+of+flight+mass+spectrometry+system+for+rapid+identification+of+bacteria+in+routine+clinical+microbiology.">Performances of the Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry system for rapid identification of bacteria in routine clinical microbiology.</a> Journal of Clinical Microbiology. 50 (8), 2568-2576 (2012).</li><li>Kawakita, T., 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=double-blind+study+of+the+safety+and+Efficacy+of+1%D-3-Hydroxybutyrate+eye+drops+for+Dry+Eye+Disease.">double-blind study of the safety and Efficacy of 1%D-3-Hydroxybutyrate eye drops for Dry Eye Disease.</a> Scientific Reports. 6, 20855 (2016).</li><li>Lee, H. 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På grund af den okulære overflades paucibakterielle tilstand har mange laboratorier haft svært ved at isolere okulære commensals7,20, hvilket resulterer i lavt antal prøver med vækst, lav overflod og lav mangfoldighed8. Denne metode forbedrer standardkulturpraksissenbetydeligt 4,21 ved at tillægge et berigelsestrin samt en nydesignet øjenpind og identifikation af MALDI-TOF ...

Formålet med denne protokol er at forbedre den specifikke isolering af levedygtige og sjældne aerobe og fakultative anaerob mikrober fra den okulære bindehinde for at karakterisere okulært mikrobiom. Omfattende undersøgelser har profileret commensal slimhinde samfund på hud, tarm, luftveje og kønsorganer og viser, at disse samfund påvirker udviklingen af immunsystemet ogrespons 1,2,3. Okulære commensal samfund har vist sig at ændre sig under visse sygdomme patologier, såsom Tør øjensygdom4, Sjögrens syndrom5 og diabetes6. Men evnen til at definere en typisk okulær overflade commensal samfund er hæmmet af deres relativt lave overflod i forhold til de andre slimhinde steder6,7,8. Dette giver anledning til en kontrovers om, hvorvidt der er en hjemmehørende okulær mikrobiom, og hvis det findes, om det adskiller sig fra huden mikrobiom og dermed dens lokale indvirkning på den medfødte immunsystemet udvikling og respons. Denne protokol kan hjælpe med at løse dette problem.
Generelt er metoder til at definere den okulære commensal niche baseret på sekventering og kulturbaserede teknikker4,7,9. 16 S rDNA sekventering og BRISK analyse7 viser en bredere mangfoldighed end kulturbaserede teknikker, men er ude af stand til at skelne mellem levende og døde mikrober. Da den okulære overflade er fjendtligt indstillet over for mange mikrober på grund af at rive filmens antimikrobielle egenskaber4 generere en lang række DNA-fragmenter, vil DNA-baserede tilgange opdage disse artefakter, som kan forvrænge dataene mod identifikation af døde bakterier som bosiddende commensals snarere end forurenende stoffer. Dette resulterer i afvigende commensal identifikation og karakterisering af okulære rum som værende højere i mikrobe overflod og mangfoldighed10. Dette gør det vanskeligt at definere det bosiddende okulære mikrobiom via DNA-baserede metoder. standardkulturbaserede teknikker er ikke i stand til at påvise commensals, fordi belastningen er for lav11. Vores metode forbedrer standardpraksis ved at bruge en tynd vatpind, der kan målrette konjunktivaen og dermed undgå forurening fra nabohud, samt konceptet om, at levedygtige organismer kan beriges af kort kultur i næringsrige tætte medier med det formål at genoplive levedygtige, men ikke-dyrkelige, samt berigelse for sjældne levedygtige mikrober.
Resultaterne, relativ overflod af okulære commensals per øjenpind, karakteriserer bindehinden hjemmehørende mikrobiom og er vigtige for komparative formål. Vores data viser, at der er forskel på hud og konjunktivalmikrobiota samt større diversitet med øget alder og en kønsspecifik forskel i overflod. Desuden har denne tilgang reproducerbart fundet commensale forskelle i knock-out mus12. Denne protokol kan anvendes til at beskrive det okulære mikrobiom, som kan variere på grund af caging praksis, geografi, eller sygdomstilstand, samt de lokale virkninger af commensal metabolitter og produkter på immunsystemet udvikling og respons.

<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

Den okulære overflade blev engang betragtet som immun privilegeret og abiotisk, men for nylig ser det ud til, at der er en lille, men vedvarende commensal tilstedeværelse. Identifikation og overvågning af bakteriearter ved okulær slimhinde har været udfordrende på grund af deres lave forekomst og begrænsede tilgængelighed af passende metoder til commensal vækst og identifikation. Der er to standard tilgange: kultur baseret eller DNA sekventering metoder. Den første metode er problematisk på grund af de begrænsede genindvindbare bakterier, og den anden tilgang identificerer både levende og døde bakterier, der fører til en afvigende repræsentation af det okulære rum. Vi udviklede en robust og følsom metode til bakteriel isolation ved at bygge videre på standard mikrobiologiske dyrkningsteknikker. Dette er en vatpind-baseret teknik, ved hjælp af en "in-lab" lavet tynd vatpind, der er målrettet den nederste bindehinde, efterfulgt af en forstærkning skridt for aerob og fakultet anaerob slægter. Denne protokol har gjort det muligt for os at isolere og identificere konjunktivalarter som Corynebacterium spp., Coagulase Negative Staphylococcus spp., Streptococcus spp.osv. Tilgangen er egnet til at definere commensal mangfoldighed hos mus under forskellige sygdomsforhold.

Repræsentative resultater for en øjenprøveplade, der demonstrerer forskellige metoder til plating, er afbilledet i figur 3A, der viser morfologisk forskellige isolater fra C57BL/6-mus. For hver særskilt isolere, kolonierne blev talt i strimlen og den relative overflod, unikke Colony Forming Units (CFUs) per øjenpind, beregnet og plottet til sammenligning formål. Til mikrobiologisk karakterisering blev bakterier plukket fra individuelle museøjepindeplader for at producere en mester TSA...

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

Conjunctival Commensal Isolation and Identification in Mice

Præsenteret her er en protokol for isolering og forstærkning af aerob og fakultativ anaerob mus conjunctival commensal bakterier ved hjælp af en unik øjenpind og kultur-baseret berigelse skridt med efterfølgende identifikation af mikrobiologiske baserede metoder og MALDI-TOF massespektrometri.

Konjunkturbestemt fællesskabsisolation og identifikation hos mus

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

Denne metode identificerer levedygtige okulære konjunktival mikroorganismer, som er udfordrende på grund af øjnene fjendtlige miljø. Det kan hjælpe med at besvare, om et okulært mikrobiom eksisterer, og hvordan bakteriel tilstedeværelse ændrer sig i øjensygdomme. I modsætning til DNA-sekventering, som identificerer både levedygtige og ikke-levedygtige mikroorganismer, identificerer denne metode kun levedygtige mikroorganismer, hvilket resulterer i en klarere forståelse af det okulære samfund.Undersøgelser tyder på, at vanskeligt at diagnosticere øjensygdomme som ikke autoimmune og autoimmune tørre øjne har unikke okulære bakteriel tilstedeværelse. Denne metode kan bruges til at diagnosticere øjensygdomme med karakteristiske mikrobielle signaturer. Begynd med autoklavering af den passende mængde bomuldsvat og tandstikkere for antallet af mus, der skal podes, og knib derefter 1/2 et centimeter langt stykke bomuldsvat og drille det ud ved at trække på kanterne for at danne et fladt enkelt porøst lag, der stopper lige før batting falder fra hinanden.Swirl batting omkring en af de skarpe ender af tandstikker ved let at holde strakt ud stykke på tandstikker spids, da det er snoet. Den færdige øjenpind vil have et meget tyndt lag bomuld strakt over spidsen strækker sig ca 1 /2 til en centimeter væk fra spidsen. Sæt svaberprøverne i et lille bægerglas, svaber side ned og dæk dem autoklaver dem.Rengør arbejdsområdet med et desinfektionsmiddel for at minimere kontaminering og aliquot 0,5 milliliter steril hjernehjerteinfusion, eller BHI-medier i mærkede 1,5 milliliter sterile mikrocentrifugerør. Hætte rørene i et stativ og sæt stativet på is. Opsæt arbejdsgangen fra venstre mod højre startende med musens bedøvelsesstation, som indeholder buret med de eksperimentelle mus, tomt sterilt bur, rumtemperaturbedøvelse, 25 gauge nål og en en milliliter sprøjte.Dernæst forberede et øje swabbing station, der indeholder aliquoted BHI på is, steriliserede øjenprøver, smøre øjendråber, biohazard container, rene papirhåndklæder, og 70% isopropanol spray. Endelig skal du oprette platingstationen med rumtemperaturblodplader, en 10 mikroliter pipette, 10 mikroliter sterile engangsspidser og en biohazard affaldsbeholder. Sørg for, at musen er korrekt bedøvet ved at klemme en bagfod pad.Fortsæt kun, hvis der ikke er nogen bevægelse. Tildel den ene hånd til at håndtere bedøvede mus og de andre hænder til at håndtere øjenprøven og kulturen. Fjern musen fra buret og læg den oven på arbejdsfladen placeret på siden med venstre øje udsat.Spray behandskede hænder med isopropanol og tør dem med et køkkenrulle. Uncap den mærkede BHI mikro centrifuge rør med dedikerede medier håndtering hånd og placere de to tilbage i rack. Dyp den bomuldsbelagte spids af øjenprøven i BHI, og træk derefter vatpinden ud af røret, mens du hvirvler spidsen to gange mod slangen for at fjerne overskydende væske og fjerne den.Hold forsigtigt musen ved nakken med musens håndteringshånd. Med den anden side, placere spidsen af øjet podning mod mediale conjunctival region af venstre øje. Tryk let på øjeæblet og flyt vatpinden i en vinduesvaskbevægelse mellem det nederste øjenlåg og øjet 10 gange, og hold konstant tryk.Uden at røre ved pelsen skal du forsigtigt fjerne spidsen af podepinden vinkelret på, hvor den blev indsat. Placer vatpinden bomuldssiden nedad, direkte ind i et mærket mikrocentrifugerør med BHI-medier. Påfør et øjendråbe på det podede øje.Hvis det ønskes, erhverve hud eller pels podninger til kontrol prøver, sterilisering handsker passende mellem hver vatpind. Når du er færdig, skal du returnere musen til buret. Lad vatpinden stå i 10 til 15 minutter på is, steriliser derefter behandskede hænder og fjern vatpinden, mens du blander spidsen i mediet i 10 rotationer.Træk vatpinden tilbage ved at hvirvle spidsen mod slangens indervæg i fem omdrejninger og bortskaffe den i en biofarlig beholder. Gentag processen for hver mus. Prøven beriges ved at inkubere røret statisk i en time ved 37 grader Celsius.Under inkubationen skal du mærke en stuetemperatur TSA-plade pr. museøjepind eller kontrolpinne og dele den i halvdelen. Fjern de berigede prøver fra inkubatoren og læg dem på is. Kort vortex prøverne til at blande, derefter aliquot 10 mikroliter af prøven på TSA plade og vippe pladen til at danne en strimmel.Gentag det to gange. På den anden side af pladerne skillelinje, skabe 10 prikker med 10 mikroliter af prøven hver. Inkubat pladerne ved 37 grader Celsius i 18 timer, to dage og fire dage i et rent kammer, der forhindrer agarplader i at tørre.Tæl kolonierne i strimlerne. Bemærk morfologi og beregn kolonidannende enheder pr. vatpind for morfologisk lignende isolater. Se på prikkerne, hvor unikke organismer ikke fanges i strimlerne.For at lette visualiseringen belagte vi bakterier tæt i midten og højre plade. Typisk observerede vi meget færre bakterier i øjenprøveplader. Bemærk, at øjenprøverne af og til ikke kan give bakterier, der kan genvindes.En repræsentativ øjenpind plade med morfologisk forskellige isolater fra en C57 sort seks mus er vist her. For hver særskilt isoleret, kolonierne blev talt i strimlen og den relative overflod blev beregnet og plottet. Til mikrobiologisk karakterisering blev bakterier plukket fra individuelle museøjepindeplader for at producere en mester TSA-plade.Da væksten dukkede op, blev der kørt yderligere tests for at karakterisere eller identificere mikroberne. Masterpladen blev brugt til at give nok inoculum til at udvide de respektive isolater. For at identificere isolater blev en TSA-plade stribet og inkuberet natten over, og MALDI-TOF MS blev udført.Eksempler på isolater vist her blev identificeret som Streptococcus acidominimus og Aerococcus viridans. Signifikant forskellige niveauer af commensal organismer blev genvundet fra mandlige og kvindelige C57 sorte seks mus. Streptococcus acidominimus, Aerococcus viridans koagulerer negative Staphylococci og E.coli blev isoleret fra C57 sorte seks N mus.Når du forsøger denne protokol, skal du huske på, at det bedste resultat opnås ved at belægge vatpinden tyndt og tage tid under øjenprøvetrinnet. Hvis adgangen til MALDI-TOF MS er begrænset, kan isolaterne identificeres ved hjælp af mikrobiologiske og biokemiske test.

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.

Isolering og analyse af Brain-sekvestrerede Leukocytter fra<em&gt; Plasmodium berghei</em&gt; ANKA-inficerede mus

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

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.

Rapid Identifikation af Gram-negative bakterier fra Blood kulturvæsken Brug MALDI-TOF-massespektrometri

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.

Isolering, identifikation og oprensning af murine thymusepitelmonolag Celler

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.

Neutrofil Isolering og analyse for at fastslå deres rolle i lymfom Cell Følsomhed til terapeutiske midler

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.

Identifikation af Plasmodesmal lokalisering sekvenser i proteiner i 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.

Isolering af Salmonella typhimurium-indeholdende Phagosomes fra makrofager

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.

Identifikation og isolering af Oligopotent og afstamning-begået myeloide ophav fra mus knoglemarv

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.

Isolering og identifikation af ekstravaskulære immunceller af hjertet

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.

En barciak Oral Administration Model til Study Commensal-Induced medfødte immunrespons

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.