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

Financiering van P30 DK034854 ondersteunde VY, LB en studies in het Massachusetts Host-Microbiome Center en financiering van NIH / NEI R01 EY022054 ondersteunde MG.

Alle procedures met muizen volgen de richtlijnen van het Institutional Animal Care and Use Committee. Volg de laboratoriumveiligheidsrichtlijnen (zoals voorgeschreven door uw afdeling Institutionele Milieuhygiëne) bij het werken met micro-organismen en mogelijk verontreinigde materialen. Gebruik geschikte afvalrecipiënten en ontsmettingsprocedures voordat mogelijk biologisch verontreinigde materialen worden verwijderd.
1. Voorbereiding van oogdoekjes, opstelling van het werkveld, uitstrijkjes ...

<ol>
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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+pathogen+or+commensal:+a+PCR+based+study+of+surface+bacterial+flora+in+normal+and+dry+eyes.">Ocular pathogen or commensal: a PCR based study of surface bacterial flora in normal and dry eyes.</a> Investigative Ophthalmology and Visual Science. 48 (12), 5616-5623 (2007).</li><li>Wang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sj&#246;gren-Like+Lacrimal+Keratoconjunctivitis+in+Germ-Free+Mice.">Sj&#246;gren-Like Lacrimal Keratoconjunctivitis in Germ-Free Mice.</a> International Journal of Molecular Sciences. 19 (2), 565-584 (2018).</li><li>Ham, B., Hwang, H. B., Jung, S. H., Chang, S., Kang, K. D., Kwon, M. 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Vanwege de paucibacteriële toestand van het oculaire oppervlak hebben veel laboratoria moeite gehad met het isoleren van oculaire commensalen7,20, wat resulteerde in een laag aantal monsters met groei, lage overvloed en lage diversiteit8. Deze methode verbetert de standaard kweekpraktijken4,21 aanzienlijk door de toevoeging van een verrijkingsstap, evenals een opnieuw ontworpen oo...

Het doel van dit protocol is om de specifieke isolatie van levensvatbare en zeldzame aerobe en facultatieve anaerobe microben uit het oculaire bindvlies te verbeteren om het oculaire microbioom te karakteriseren. Uitgebreide studies hebben commensale mucosale gemeenschappen geprofileerd op de huid, darmen, luchtwegen en geslachtsorganen en tonen aan dat deze gemeenschappen de ontwikkeling van het immuunsysteem en de respons1,2,3beïnvloeden . Van oculaire commensale gemeenschappen is aangetoond dat ze veranderen tijdens bepaalde ziektepathologieën, zoals droge ogenziekte4,syndroom van Sjogren5 en diabetes6. Toch wordt het vermogen om een typische oculaire oppervlaktecommensale gemeenschap te definiëren belemmerd door hun relatief lage overvloed in vergelijking met de andere mucosale sites6,7,8. Dit leidt tot een controverse over de vraag of er een ingezeten oculaire microbioom is en of het bestaat, of het verschilt van het huidmicrobioom en bijgevolg het lokale effect ervan op de ontwikkeling en reactie van het aangeboren immuunsysteem. Dit protocol kan helpen deze vraag op te lossen.
Over het algemeen zijn benaderingen om de oculaire commensale niche te definiëren gebaseerd op sequencing en op cultuur gebaseerde technieken4,7,9. S rDNA-sequencing en BRISK-analyse7 vertonen een bredere diversiteit dan op cultuur gebaseerde technieken, maar kunnen geen onderscheid maken tussen levende en dode microben. Omdat het oculaire oppervlak vijandig is voor veel microben als gevolg van de antimicrobiële eigenschappen van traanfilm4 die een grote reeks DNA-fragmenten genereren, zullen DNA-gebaseerde benaderingen deze artefacten detecteren die de gegevens kunnen scheeftrekken in de richting van identificatie van dode bacteriën als ingezeten commensalen in plaats van verontreinigingen. Dit resulteert in afwijkende commensale identificatie en karakterisering van de oculaire ruimte als hoger in microbenrijkdom en diversiteit10. Dit maakt het moeilijk om het ingezeten oculaire microbioom te definiëren via DNA-gebaseerde methoden. Overwegende dat standaard kweekgebaseerde technieken geen commensalen kunnen detecteren omdat de belasting te laag is11. Onze methode verbetert de standaardpraktijken door een dun wattenstaafje te gebruiken dat zich op het bindvlies kan richten, waardoor besmetting van de naburige huid wordt vermeden, evenals het concept dat levensvatbare organismen kunnen worden verrijkt door een korte cultuur in voedingsrijke media met als doel levensvatbare maar niet-cultureerbare, evenals verrijking voor zeldzame levensvatbare microben te reanimeren.
De resultaten, relatieve overvloed aan oculaire commensalen per oogdoekje, karakteriseren het conjunctiva resident microbioom en zijn belangrijk voor vergelijkende doeleinden. Onze gegevens tonen aan dat er een verschil is tussen huid en conjunctivale microbiota, evenals een grotere diversiteit met verhoogde leeftijd en een geslachtsspecifiek verschil in overvloed. Bovendien heeft deze aanpak reproduceerbaar commensale verschillen gevonden in knock-out muizen12. Dit protocol kan worden toegepast om het oculaire microbioom te beschrijven dat kan variëren als gevolg van kooipraktijken, geografie of ziektetoestand, evenals de lokale effecten van commensale metabolieten en producten op de ontwikkeling en respons van het immuunsysteem.

<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

Het oculaire oppervlak werd ooit beschouwd als immuun bevoorrecht en abiotisch, maar onlangs lijkt het erop dat er een kleine, maar aanhoudende commensale aanwezigheid is. De identificatie en monitoring van bacteriële soorten in het oogslijmvlies waren een uitdaging vanwege hun geringe overvloed en beperkte beschikbaarheid van geschikte methodologie voor commensale groei en identificatie. Er zijn twee standaardbenaderingen: op cultuur gebaseerde of DNA-sequencingmethoden. De eerste methode is problematisch vanwege de beperkte herwinbare bacteriën en de tweede benadering identificeert zowel levende als dode bacteriën die leiden tot een afwijkende weergave van de oculaire ruimte. We ontwikkelden een robuuste en gevoelige methode voor bacteriële isolatie door voort te bouwen op standaard microbiologische kweektechnieken. Dit is een op uitstrijkjes gebaseerde techniek, met behulp van een "in-lab" gemaakt dun wattenstaafje dat zich richt op het onderste bindvlies, gevolgd door een versterkingsstap voor aerobe en facultatieve anaerobe geslachten. Dit protocol heeft ons in staat gesteld om conjunctivale soorten zoals Corynebacterium spp., Coagulase Negative Staphylococcus spp., Streptococcus spp., enz. te isoleren en te identificeren. De aanpak is geschikt om commensale diversiteit bij muizen onder verschillende ziekteomstandigheden te definiëren.

Representatieve resultaten voor een oogswabplaat die verschillende methoden voor plateren aantoont, zijn afgebeeld in figuur 3A met morfologisch diverse isolaten van C57BL/6-muis. Voor elk afzonderlijk isolaat werden de kolonies geteld in de strip en de relatieve overvloed, unieke Kolonievormende Eenheden (CFUs) per oogswab, berekend en uitgezet voor vergelijkingsdoeleinden. Voor microbiologische karakterisering werden bacteriën uit individuele muisoogswabplaten geplukt om een master TSA-pl...

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

Conjunctival Commensal Isolation and Identification in Mice

Hier wordt een protocol gepresenteerd voor de isolatie en versterking van aerobe en facultatieve anaerobe muisconjunctivale commensale bacteriën met behulp van een uniek oogswab en op cultuur gebaseerde verrijkingsstap met daaropvolgende identificatie door microbiologische methoden en MALDI-TOF massaspectrometrie.

Conjunctivale commensale isolatie en identificatie bij muizen

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

Deze methode identificeert levensvatbare oculaire conjunctivale micro-organismen, wat een uitdaging is vanwege de vijandige omgeving van de ogen. Het kan helpen beantwoorden of een oculaire microbioom bestaat en hoe bacteriële aanwezigheid verandert in oogaandoeningen. In tegenstelling tot DNA-sequencing, die zowel levensvatbare als niet-levensvatbare micro-organismen identificeert, identificeert deze methode alleen levensvatbare micro-organismen, wat resulteert in een duidelijker begrip van de oculaire commensale gemeenschap.Studies suggereren dat moeilijk te diagnosticeren oogziekten zoals niet-auto-immuun en auto-immuun droge ogen unieke oculaire bacteriële aanwezigheid hebben. Deze methode kan worden gebruikt om oogziekten te diagnosticeren met onderscheidende microbiële handtekeningen. Begin met het autoclaaf van de juiste hoeveelheid katoenen batting en tandenstokers voor het aantal muizen dat moet worden uitgewist, knijp vervolgens 1/2 een centimeter lang stuk katoenen batting af en plaag het door aan de randen te trekken om een vlakke enkele poreuze laag te vormen die stopt net voordat het slaan uit elkaar valt.Wervel het slaan rond een van de scherpe uiteinden van de tandenstoker door het uitgerekte stuk lichtjes op de tandenstokerpunt te houden terwijl het gedraaid is. Het afgewerkte oogdoekje heeft een zeer dunne laag katoen die over de punt is uitgerekt en zich ongeveer 1/2 tot een centimeter van de punt uitstrekt. Steek de wattenstaafjes in een klein bekerglas, veeg ze met de andere kant naar beneden en dek ze af en autoclaveer ze.Reinig het werkgebied met een ontsmettingsmiddel om besmetting te minimaliseren en voer 0,5 milliliter steriele hersenhartinfusie of BHI-media in gelabelde steriele microcentrifugebuizen van 1,5 milliliter. Sluit de buizen af in een rek en zet het rek op ijs. Stel de workflow in van links naar rechts, te beginnen met het anesthesiestation van de muis, dat de kooi bevat met de experimentele muizen, lege steriele kooi, anesthesie op kamertemperatuur, een naald van 25 gauge en een spuit van één milliliter.Bereid vervolgens een oogdoekstation voor dat de aliquoted BHI op ijs, gesteriliseerde oogswabs, smerende oogdruppels, biogevaarlijke container, schone papieren handdoeken en 70% isopropanolspray bevat. Zet ten slotte het beplatingsstation op met bloedagarplaten op kamertemperatuur, een pipet van 10 microliter, steriele wegwerptips van 10 microliter en een biogevaarlijke afvalcontainer. Zorg ervoor dat de muis goed wordt verdoofd door in een achtervoetkussen te knijpen.Ga alleen verder als er geen beweging is. Wijs één hand toe om verdoofde muizen te behandelen en de andere handen om het oogswab en de cultuur te hanteren. Haal de muis uit de kooi en plaats deze bovenop het werkoppervlak dat op zijn kant is geplaatst terwijl het linkeroog wordt blootgesteld.Spuit gehandschoende handen met isopropanol en droog ze met een papieren handdoek. Ontscap de gelabelde BHI microcentrifugebuis met de speciale mediahandgreep en plaats de twee terug in het rek. Dompel de met katoen beklede punt van het oogdoekje in de BHI en trek het wattenstaafje uit de buis terwijl u de punt twee keer tegen de binnenband wervelt om overtollige vloeistof te verwijderen en te verwijderen.Houd de muis met de hand voorzichtig bij het nekvel. Plaats met de andere hand de punt van het oogdoekje tegen het mediale conjunctivale gebied van het linkeroog. Druk de oogbol licht in en beweeg het wattenstaafje in een raamwasbeweging tussen het onderste ooglid en het oog 10 keer, met constante druk.Zonder de vacht aan te raken, verwijdert u voorzichtig de punt van het wattenstaafje loodrecht op waar het is ingebracht. Plaats het wattenstaafje met de wattenstaafjezijde naar beneden, direct in een gelabelde microcentrifugebuis met BHI-media. Breng een oogdruppel aan op het uitgewten oog.Neem indien gewenst huid- of pelsdoekjes voor controlemonsters en steriliseer handschoenen op de juiste manier tussen elk wattenstaafje. Als u klaar bent, keert u de muis terug naar de kooi. Laat het wattenstaafje 10 tot 15 minuten op ijs staan, steriliseer vervolgens gehandschoende handen en verwijder het wattenstaafje terwijl u de punt gedurende 10 omwentelingen in het medium mengt.Trek het wattenstaafje terug door de punt vijf keer tegen de binnenwand van de buis te draaien en gooi het weg in een biogevaarlijke container. Herhaal het proces voor elke muis. Verrijk het monster door de buis een uur statisch te incuberen bij 37 graden Celsius.Label tijdens de incubatie één TSA-plaat op kamertemperatuur per muisoogdoekje of controledoekje en verdeel het in tweeën. Haal de verrijkte monsters uit de incubator en leg ze op ijs. Draai de monsters kort om te mengen, aliquot vervolgens 10 microliter van het monster op de TSA-plaat en kantel de plaat om een strip te vormen.Herhaal dit twee keer. Maak aan de andere kant van de scheidingslijn 10 stippen met elk 10 microliter monster. Incubeer de platen gedurende 18 uur, twee dagen en vier dagen op 37 graden Celsius in een schone kamer die voorkomt dat agarplaten drogen.Tel de kolonies in de stroken. Noteer morfologie en bereken kolonievormende eenheden per wattenstaafje voor morfologisch vergelijkbare isolaten. Kijk naar de stippen waar unieke organismen niet worden gevangen in de stroken.Om de visualisatie te vergemakkelijken, hebben we bacteriën dicht in de middelste en rechterplaten geplateerd. Meestal zagen we veel minder bacteriën in oogswabplaten. Merk op dat de oogdoekjes soms geen herwinbare bacteriën opleveren.Een representatieve oogswabplaat met morfologisch diverse isolaten van een C57 zwarte zesmuis wordt hier getoond. Voor elk afzonderlijk isolaat werden de kolonies in de strook geteld en werd de relatieve overvloed berekend en uitgezet. Voor microbiologische karakterisering werden bacteriën uit individuele muisoogswabplaten geplukt om een master TSA-plaat te produceren.Toen de groei verscheen, werden aanvullende tests uitgevoerd om de microben te karakteriseren of te identificeren. De hoofdplaat werd gebruikt om voldoende entmateriaal te leveren om de respectieve isolaten uit te breiden. Om isolaten te identificeren, werd een TSA-plaat 's nachts gestreept en geïncubeerd en werd MALDI-TOF MS uitgevoerd.Voorbeelden van hier getoonde isolaten werden geïdentificeerd als Streptococcus acidominimus en Aerococcus viridans. Significant verschillende niveaus van commensale organismen werden teruggevonden van mannelijke en vrouwelijke C57 zwarte zes muizen. Streptococcus acidominimus, Aerococcus viridans coagulates negatieve Stafylokokken en E.coli werden geïsoleerd uit C57 zwarte zes N muizen.Houd er bij het proberen van dit protocol rekening mee dat het beste resultaat wordt bereikt door het wattenstaafje dun te coaten en tijd te nemen tijdens de ooguitstrijkstap. Als de toegang tot MALDI-TOF MS beperkt is, kunnen de isolaten worden geïdentificeerd door microbiologische en biochemische tests.

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.

Isolatie en analyse van Brain-afgezonderd Leukocyten uit<em&gt; Plasmodium berghei</em&gt; ANKA-geïnfecteerde muizen

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.

Snelle identificatie van Gram-negatieve bacteriën van Blood Culture Bouillon Met behulp van MALDI-TOF massaspectrometrie

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.

Isolatie, identificatie en zuivering van Muriene thymus epitheelcellen

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.

Neutrofielen Isolatie en analyse van hun rol in de Lymphoma Cell Gevoeligheid voor therapeutische middelen te bepalen

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.

Identificatie van Plasmodesmal lokalisatie sequenties in eiwitten 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.

Isolatie van Salmonella typhimurium-met Phagosomes van Macrophages

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.

Identificatie en isolatie van de Oligopotent en Lineage-begaan myeloïde voorlopercellen uit beenmerg van de muis

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.

Isolatie en identificatie van Extravascular immuuncellen van het hart

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.

Een mondelinge beheermodel voor wasmot te Study Commensal-Induced aangeboren immuunresponsen

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.