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

Støtte fra P30 DK034854 støttet VY, LB og studier i Massachusetts Host-Microbiome Center og finansiering fra NIH/NEI R01 EY022054 støttet MG.

Alle prosedyrer som involverer mus følger retningslinjene fra Institusjonelt dyrepleie- og bruksutvalg. Følg laboratoriets sikkerhetsretningslinjer (som anvist av din institusjonelle miljøhelse- og sikkerhetsavdeling) når du arbeider med mikroorganismer og potensielt forurensede materialer. Bruk egnede avfallsbeholdere og dekontamineringsprosedyrer før avhending av potensielt biofarlige forurensede materialer.
1. Forberedelse av øyepinne, oppsett av arbeidsfelt, museøyeswabbing og prøveb...

<ol>
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På grunn av den paucibakterielle tilstanden til okulær overflate har mange laboratorier hatt problemer med å isolere okulære commensals7,20, noe som resulterer i lavt antall prøver med vekst, lav overflod og lavt mangfold8. Denne metoden forbedrer standard kulturpraksisbetydelig 4,21 ved å legge til et berikelsestrinn, samt en redesignet øyepinne og identifikasjon av MALDI-T...

Målet med denne protokollen er å forbedre spesifikk isolasjon av levedyktige og sjeldne aerobe og fakultets anaerobe mikrober fra okulær konjunktiv for å karakterisere okulær mikrobiomet. Omfattende studier har profilert commensal mucosal samfunn på hud, tarm, luftveier og kjønnsorganer og viser at disse samfunnene påvirker utviklingen av immunsystemet og responsen1,2,3. Okulære commensale samfunn har vist seg å endre seg under visse sykdomspatologier, for eksempel Tørr øyesykdom4, Sjogrens syndrom5 og diabetes6. Likevel er evnen til å definere et typisk okulært overflatekompmensalt samfunn hemmet av deres relativt lave overflod sammenlignet med de andre mucosalstedene6,7,8. Dette fører til en kontrovers om hvorvidt det er et bosatt okulært mikrobiom og om det eksisterer, om det er forskjellig fra hudens mikrobiom og følgelig den lokale effekten på den medfødte immunsystemutviklingen og responsen. Denne protokollen kan bidra til å løse dette spørsmålet.
Generelt er tilnærminger for å definere okulær commensal nisje basert på sekvensering og kulturbaserte teknikker4,7,9. 16 S rDNA-sekvensering og BRISK-analyse 7 viser etbredere mangfold enn kulturbaserte teknikker, men klarer ikke å skille mellom levende og døde mikrober. Siden den okulære overflaten er fiendtlig innstilt til mange mikrober på grunn av tårefilmens antimikrobielle egenskaper4 som genererer et stort utvalg av DNA-fragmenter, vil DNA-baserte tilnærminger oppdage disse artefaktene som kan forskyve dataene mot identifisering av døde bakterier som bosatte commensals i stedet for forurensninger. Dette resulterer i avvikende commensal identifikasjon og karakterisering av okulærrommet som høyere i mikrobe overflod og mangfold10. Dette gjør det vanskelig å definere det beboende okulære mikrobiomet via DNA-baserte metoder. Standard kulturbaserte teknikker kan imidlertid ikke oppdage commensals fordi belastningen er for lav11. Vår metode forbedrer standardpraksis ved å bruke en tynn vattpinne som kan målrette konjunktivene, og dermed unngå forurensning fra nærliggende hud, samt konseptet om at levedyktige organismer kan berikes av kort kultur i næringstett media med mål om å gjenopplive levedyktige, men ikke-kultiverbare, samt berike for sjeldne levedyktige mikrober.
Resultatene, relativ overflod av okulære commensals per øyepinne, karakteriserer konjunktiv bosatt mikrobiom og er viktige for komparative formål. Våre data viser at det er forskjell på hud og konjunktiv mikrobiota, samt større mangfold med økt alder og en kjønnsspesifikk forskjell i overflod. Videre har denne tilnærmingen reprodusert funnet commensal forskjeller i knock-out mus12. Denne protokollen kan brukes til å beskrive okulært mikrobiom som kan variere på grunn av caging praksis, geografi eller sykdom tilstand, samt lokale effekter av commensal metabolitter og produkter på immunsystem utvikling 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 overflaten ble en gang ansett som immunprivilegert og abiotisk, men nylig ser det ut til at det er en liten, men vedvarende commensal tilstedeværelse. Identifisering og overvåking av bakteriearter ved okulær slimhinne har vært utfordrende på grunn av deres lave overflod og begrensede tilgjengelighet av passende metodikk for commensal vekst og identifisering. Det er to standard tilnærminger: kulturbaserte eller DNA-sekvenseringsmetoder. Den første metoden er problematisk på grunn av de begrensede utvinnbare bakteriene, og den andre tilnærmingen identifiserer både levende og døde bakterier som fører til en avvikende representasjon av okulærrommet. Vi utviklet en robust og sensitiv metode for bakteriell isolasjon ved å bygge videre på standard mikrobiologiske kultiveringsteknikker. Dette er en vattpinnebasert teknikk, ved hjelp av en "in-lab" laget tynn vattpinne som retter seg mot nedre konjunktiv, etterfulgt av et forsterkningstrinn for aerob og fakultets anaerob slekt. Denne protokollen har gjort det mulig for oss å isolere og identifisere konjunktivale arter som Corynebacterium spp., Coagulase Negative Staphylococcus spp., Streptococcus spp., etc. Tilnærmingen er egnet til å definere commensal mangfold hos mus under forskjellige sykdomstilstander.

Representative resultater for en øyepinneplate som demonstrerer forskjellige metoder for plating er avbildet i figur 3A som viser morfologiske forskjellige isolasjoner fra C57BL / 6 mus. For hver distinkt isolasjon ble koloniene telt i stripen og den relative overfloden, unike Colony Forming Units (CFUer) per øyepinne, beregnet og plottet for sammenligningsformål. For mikrobiologisk karakterisering ble bakterier plukket fra individuelle museøyepinneplater for å produsere en master TSA-p...

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

Conjunctival Commensal Isolation and Identification in Mice

Presentert her er en protokoll for isolering og forsterkning av aerob og fakultets anaerob mus konjunktivale kommensale bakterier ved hjelp av en unik øyepinne og kulturbasert berikelse trinn med påfølgende identifikasjon av mikrobiologiske baserte metoder og MALDI-TOF masse spektrometri.

Konjunktival commensal isolasjon og identifikasjon 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 metoden identifiserer levedyktige okulære konjunktivale mikroorganismer, noe som er utfordrende på grunn av øynene fiendtlig miljø. Det kan bidra til å svare på om et okulært mikrobiom eksisterer og hvordan bakteriell tilstedeværelse endres i øyesykdom. I motsetning til DNA-sekvensering, som identifiserer både levedyktige og ikke-levedyktige mikroorganismer, identifiserer denne metoden bare levedyktige mikroorganismer, noe som resulterer i en klarere forståelse av okulære commensal samfunnet.Studier tyder på at vanskelig å diagnostisere øyesykdommer som ikke autoimmun og autoimmun tørt øye har unik okulær bakteriell tilstedeværelse. Denne metoden kan brukes til å diagnostisere øyesykdommer med særegne mikrobielle signaturer. Begynn med å autoklavere riktig mengde bomullsslag og tannpirkere for antall mus som skal swabbes, og klem deretter av 1/2 et centimeter langt stykke bomullsslag og ert det ut ved å trekke på kantene for å danne et flatt enkelt porøst lag som stopper like før battingen faller fra hverandre.Virvle batting rundt en av de skarpe endene av tannpirkeren ved å holde lett den utstrakte delen på tannpirkerspissen som den er vridd. Den ferdige øyepinnen vil ha et veldig tynt lag bomull strukket over spissen som strekker seg omtrent 1/2 til en centimeter unna spissen. Sett vattpinnene inn i et lite beger, vattpinnesiden ned og dekk og autoklaver dem automatisk.Rengjør arbeidsområdet med et desinfeksjonsmiddel for å minimere forurensning og aliquot 0,5 milliliter steril hjernehjerteinfusjon, eller BHI-medier i merkede 1,5 milliliter sterile mikrosentrifugerør. Hette rørene i et stativ og sett stativet på is. Sett opp arbeidsflyt fra venstre til høyre fra og med musens bedøvelsesstasjon, som inneholder buret med eksperimentelle mus, tomt sterilt bur, romtemperaturbedøvelse, 25 gauge nål og en en milliliter sprøyte.Deretter forbereder du en øyeswabbingstasjon som inneholder den alisiterte BHI på is, steriliserte øyepinner, smørende øyedråper, biofarebeholder, rene papirhåndklær og 70% isopropanolspray. Til slutt setter du opp platingstasjonen med romtemperatur blod agarplater, en 10 mikroliterpipette, 10 mikroliter sterile engangsspisser og en biofare avfallsbeholder. Forsikre deg om at musen er riktig bedøvet ved å klemme en bakre fotpute.Bare fortsett hvis det ikke er noen bevegelse. Be den ene hånden om å håndtere bedøvede mus og de andre hendene for å håndtere øyepinnen og kulturen. Fjern musen fra buret og plasser den på toppen av arbeidsflaten plassert på siden med venstre øye utsatt.Spray hanskede hender med isopropanol og tørk dem med et papirhåndkle. Løsne det merkede BHI mikrosentrifugerøret med den dedikerte mediehåndteringshånden og legg de to tilbake i stativet. Dypp den bomullsbelagte spissen av øyepinnen i BHI, trekk deretter vattpinnen ut av røret mens du virvler spissen to ganger mot det indre røret for å fjerne overflødig væske og fjerne den.Med musens håndteringshånd holder du forsiktig musen ved halsen. Med den annen side, plasser spissen av øyepinnen mot medial konjunktivalområdet i venstre øye. Trykk øyebollet lett ned og beveg vattpinnen i en vindusvaskbevegelse mellom nedre øyelokk og øye 10 ganger, og opprettholde konstant trykk.Uten å berøre pelsen, fjern forsiktig spissen av vattpinnen vinkelrett på hvor den ble satt inn. Plasser vattpinne bomullssiden ned, direkte inn i et merket mikro sentrifugerør med BHI-medier. Påfør en øyedråpe på det vattne øyet.Om ønskelig, skaff hud- eller pelspinner for kontrollprøver, sterilisere hansker riktig mellom hver vattpinne. Når du er ferdig, returner musen til buret. La vattpinnen stå i 10 til 15 minutter på isen, steriliser deretter hanskede hender og fjern vattpinnen mens du blander spissen i mediet i 10 rotasjoner.Trekk vattpinnen ved å virvle spissen mot den indre veggen av røret i fem rotasjoner og kast den i en biofarebeholder. Gjenta prosessen for hver mus. Berik prøven ved å inkubere røret statisk i en time ved 37 grader Celsius.Under inkubasjon, merk en romtemperatur TSA-plate per museøyepinne eller kontrollpinne og del den i halvparten. Fjern de berikede prøvene fra inkubatoren og legg dem på is. Virvel prøvene kort for å blande, deretter aliquot 10 mikroliter av prøven på TSA-platen og vipp platen for å danne en stripe.Gjenta dette to ganger. På den andre siden av platene som deler linjen, lag 10 prikker med 10 mikroliter prøve hver. Inkuber platene ved 37 grader Celsius i 18 timer, to dager og fire dager i et rent kammer som forhindrer agarplater i å tørke.Tell koloniene i strimlene. Legg merke til morfologi og beregn kolonidannende enheter per vattpinne for morfologisk lignende isolasjoner. Se på prikkene der unike organismer ikke fanges i strimlene.For å lette visualiseringen belagte vi bakterier tett i midtre og høyre plater. Vanligvis observerte vi mye færre bakterier i øyepinneplater. Vær oppmerksom på at øyepinnene noen ganger kanskje ikke gir utvinnbare bakterier.En representativ øyepinneplate med morfologisk forskjellige isolerer fra en C57 svart seks mus er vist her. For hver distinkt isolasjon ble koloniene talt i stripen og den relative overfloden ble beregnet og plottet. For mikrobiologisk karakterisering ble bakterier plukket fra individuelle museøyepinneplater for å produsere en master TSA-plate.Da veksten dukket opp, ble ytterligere tester kjørt for å karakterisere eller identifisere mikrober. Hovedplaten ble brukt til å gi nok inokulum til å utvide de respektive isolasjonene. For å identifisere isolasjoner ble en TSA-plate strødd og inkubert over natten, og MALDI-TOF MS ble utført.Eksempler på isolasjoner vist her ble identifisert som Streptococcus acidominimus og Aerococcus viridans. Betydelig forskjellige nivåer av kommensale organismer ble gjenopprettet fra mannlige og kvinnelige C57 svarte seks mus. Streptococcus acidominimus, Aerococcus viridans koagulerer negative Staphylococci og E.coli ble isolert fra C57 svart seks N mus.Når du prøver denne protokollen, må du huske på at det beste resultatet oppnås ved å belegge vattpinnen tynt og ta tid under øyepinnetrinnet. Hvis tilgangen til MALDI-TOF MS er begrenset, kan isolasjonene identifiseres ved mikrobiologisk og biokjemisk testing.

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 av Brain-sequestered Leukocytter fra<em&gt; Plasmodium berghei</em&gt; ANKA-infiserte 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 Identifikasjon av gramnegative bakterier fra blodkultur buljong Bruke 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.

Isolasjon, Identifikasjon, og rensing av Muse thymisk Epitelceller

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.

Nøytrofile Isolering og analyse for å fastslå deres rolle i lymfom Cell Følsomhet for 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.

Identifikasjon av 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.

Isolasjon av Salmonella typhimurium-som inneholder 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.

Identifisering og isolering av Oligopotent og avstamning forpliktet myelogen Progenitors fra musen benmarg

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.

Isolasjon og identifisering av ekstravaskulær overvåking av immunceller av 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.

Galleria mellonella muntlig administrasjonsmodell til Study Commensal-Induced medfødte immunreaksjoner

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.