Name: detection and enrichment of rare antigen-specific b cells for analysis of phenotype and function
Uploaded: 2017-02-16T15:00:00
Description: Watch this Scientific Journal Video about Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function at JoVE.com

This work was supported by grants from the JDRF (1-2008-994, 27-2012-450) and the National Institutes of Health (R01DK096492-05, R21AI124488-01, T32OD012201, and F30OD021477).

1. Isolering af Humane PBMC&#39;er <ol><li> Saml 30-50 ml blod ved hjælp af hepariniseret blod indsamling rør. BEMÆRK: Den mængde blod, der anvendes afhænger af den særlige eksperimentelle spørgsmål og frekvens i blod af antigenspecifikke B-celler af interesse. Hepariniseret blod kan forarbejdes straks eller vippes blidt natten over ved stuetemperatur til behandling den følgende dag. Forsinkelsen i forarbejdningen har meget lille virkning på effektiviteten af ​​berigelse og er forbundet med minima...

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Her beskriver vi en hidtil ukendt metode til at opnå isolering og berigelse af antigen-bindende B-celler fra humant perifert blod. Fremgangsmåden er let anvendelig til mus og til andre væv, såsom milten og lymfeknuderne, og er kompatibel med post-berigelse analyse af cellefænotype og funktion (manuskript under udarbejdelse). Brugeren skal være bekendt med en række variabler, som kan påvirke succes af denne procedure. Af erfaring døde celler tendens til at klæbe til de magnetiske pe...

Begrænsende fortynding analyser af antistof-secernerende celle forstadieforekomst har antydet, at B-celler reaktive mod et bestemt antigen typisk sted med en frekvens på 0,05 til 0,005% i det normale repertoire, afhængigt af status vaccination og størrelse / antal epitoper er til stede på antigenet. Den lavfrekvente af disse celler har gjort det vanskeligt at studere ændringer i deres status under udviklingen af ​​immunreaktioner, såsom efter vaccination eller udsættelse for et fremmed antigen, eller under udviklingen af ​​autoimmunitet. Tidligere har forskere gennemført isolering af antigen-reaktive B-celler under anvendelse af teknikker, der spænder fra antigen coatede plader eller kolonne adsorbenter, til antigen-coatede røde blodlegemer nulstilling, til fluorescens-aktiveret cellesortering 1, 2, 3, 4, 5, 6. though disse teknikker har haft succes med at identificere og isolere antigen-reaktive B-celler, har resultaterne varieret i form af udbytte, renhed og skalerbarhed. For nylig har vi udviklet en ny fremgangsmåde til både påvisning og berige sjældne B lymfocyt subpopulationer anvendelse af magnetiske nanopartikler. Metoden muliggør berigelse med relativt højt udbytte og renhed fra store udgangspunkter befolkninger, og er kompatibel med analyse af reaktioner på antigen. Ved at berige fra populationer af celler i suspension, hvilken fremgangsmåde eliminerer problemer, der er forbundet med geometrien af ​​antigen-coatede plader eller kolonner og begrænser gennemløb. Endelig eftersom berigede celler forblive forbundet med antigen og en fluorescerende reporter, kan de renses yderligere ved FACS-sortering. Som beskrevet heri har vi anvendt denne metode til undersøgelse af perifere blod tetanustoxoid-reaktive B-celler før og efter immunisering af humane individer, såvel som autoantigene reaktive B-celler fra individer med forskellige autoimmune lidelser, herunder type 1-diabetes, Graves &#39;sygdom, og Hashimotos sygdom 7. Fremgangsmåden fungerer lige godt i mus og menneske, og er kompatibel med analyse af antigen-reaktive B-celler fra en række forskellige væv (manuskript under udarbejdelse). I sin grundlæggende format, er perifert blod mononukleære celler først inkuberet med biotinyleret antigen sammen med antistoffer mod celleoverfladeantigener kræves til fænotypisk analyse. Dette trin mærkning efterfulgt af vask og fiksering, og tilsætning af streptavidin koblet til langt-rød-fluorescerende farvestof til detektering af biotinylerede-antigenbindende celler (figur 1). Tidligere undersøgelser har identificeret antigenspecifikke B-celler på lignende måde men under anvendelse antigener direkte konjugeret til et fluorochrom 8, 9, 10, 11. Selv om dette er en værdigtilgang, anvendelse af biotinylerede antigener i sammenhæng med streptavidin muliggør en større signalforstærkning (dermed bedre differentiering af bindende og ikke-bindende celler), især når antigener er små 12, 13, 14. En yderligere overvejelse er anvendelsen af ​​streptavidin i stedet for avidin fordi streptavidin deglycosyleret, faldende ikke-specifik binding. Yderligere bruger vi langt-rød-fluorescerende farvestof som fluorchrom grundet dens fotostabilitet, kvantumudbytte (lysstyrke), og dens lille størrelse (~ 1,3 kD). Protein-fluorochromer, såsom phycoerythrin (~ 250 kD) og allophycocyanin (~ 105 kD) 15 er ikke optimale, da de potentielt indeholde mange antigene epitoper. Anvendelse af et lille organisk fluorescerende farvestof sammensat af en enkelt epitop, såsom langtrækkende rød-fluorescerende farvestof, reducerer kompleksiteten af ​​det isolerede cellepopulation. Når celler biotinyleres-antigen og langt-rød-fluorescerende farvestof-streptavidin adsorberet, er de berigede anvendelse af anti-far-rød-fluorescerende farvestof-konjugeret magnetiske nanopartikler. Enkelte nanopartikler er ikke opdaget af de fleste flowcytometre og skal derfor ikke fjernes før rensning ved FACS sortering og downstream analyser 16. Magnetisk selektion for antigenspecifikke B-celler beriger populationen af ​​interesse, hvilket eliminerer den tid og omkostninger til sortering sjældne begivenheder ved hjælp et flowcytometer. Nedenfor viser vi repræsentative resultater fra berigelse af stivkrampe-toxoid-specifikke B-celler fra et emne før og syv dage efter tetanustoksoid booster immunisering. Vi valgte denne særlige anvendelse som et eksempel for at demonstrere evnen af denne fremgangsmåde til at berige antigenspecifikke B-celler efter akut in vivo-stimulering. Når kombineret med flowcytometri, denne metode er stand til at berige og differentierende antigen-specifik naive, hukommelse, ogplasmablast B-celler og gør det muligt for forskeren at følge ændringer i deres frekvens over tid. Derudover inkluderer vi en anden mulig nedstrøms assay, fx en ELISPOT-analyse, der viser, at celler bevarer evnen til at udskille antistof efter berigelse. En anden anvendelse af denne metode kan indebære adoptiv overførsel af berigede celler i en vært. Vi har tidligere vist celler opretholder evnen til at virke som antigenpræsenterende celler til antigenspecifikke T-celler efter isolering og overførsel (data ikke vist). Derfor er der en række mulige downstream assays, der kunne kobles til den metode, der tilsammen informerer forståelse af antigen-specifikt immunrespons. Vi har beskrevet nedenstående metode, herunder kontrol for at bestemme samlede udbytte, renhed, celle specificitet, og fold-berigelse.

<table border="0" cellpadding="0" cellspacing="0" width="594">
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	<tbody>
		<tr height="99">
			<td height="99" style="height:99px;width:164px;">Antigen of interest</td>
			<td style="width:104px;">variable</td>
			<td style="width:131px;">variable</td>
			<td style="width:196px;">At least 100 &#956;g to biotinylate easily; if using protein try to use protein that has been validated by ELISA. It must be carrier&#160; protein free.</td>
		</tr>
		<tr height="118">
			<td height="118" style="height:118px;width:164px;">Biotin for labeling; e.g. EZ link Sulfo-NHS-LC-Biotin</td>
			<td style="width:104px;">e.g. Thermo Scientific</td>
			<td style="width:131px;">e.g. 21335</td>
			<td style="width:196px;">Biotin is available in different formulations, such as those containing&#160; various length spacers, so the type used should be determined by the researcher</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Streptavidin-Alexa Fluor 647</td>
			<td style="width:104px;">Invitrogen</td>
			<td style="width:131px;">S21374</td>
			<td style="width:196px;">Can obtain from other suppliers.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:164px;">Anti-Cy5/Anti-Alexa Fluor 647 Microbeads</td>
			<td style="width:104px;">Miltenyi Biotech</td>
			<td style="width:131px;">130-091-395</td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:164px;">LS Columns</td>
			<td style="width:104px;">Miltenyi Biotech</td>
			<td style="width:131px;">130-042-401</td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:164px;">MACS manual separators</td>
			<td style="width:104px;">Miltenyi Biotech</td>
			<td style="width:131px;">variable</td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:164px;">Formaldehyde</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;">Dilute to 2% with PBS; optional if downstream assay requires live cells</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:164px;">PBS without calcium and magnesium</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:164px;">Ficoll-Paque PLUS</td>
			<td style="width:104px;">GE Healthcare</td>
			<td style="width:131px;">17-1440-02</td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="59">
			<td height="59" style="height:59px;width:164px;">Whole blood in heparinized collection tubes</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:164px;">FACS buffer (PBS + 1% BSA + 0.01% sodium azide)</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:164px;">Separation buffer (PBS + 0.5% BSA + 2mM EDTA)</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:164px;">50 mL conical tubes</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:164px;">15 mL conical tubes</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:164px;">1.5 mL Eppendorf tubes</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="96">
			<td height="96" style="height:96px;width:164px;">Surface marker&#160; reactive antibodies, Fc Block, live/dead discriminating stain, if needed</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:164px;">ELISPOT supplies, if needed</td>
			<td style="width:104px;"></td>
			<td style="width:131px;"></td>
			<td style="width:196px;"></td>
		</tr>
	</tbody>
</table>

detection and enrichment of rare antigen-specific b cells for analysis of phenotype and function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

Analyse af renhed, udbytte, og fold-berigelse ved hjælp af flowcytometri Populationer beriget som beskrevet ovenfor uundgåeligt indeholde kontaminerende celler, som ikke er bundet streptavidin- langt-rød-fluorescerende farvestof, men er fanget i matrixen. Disse urenheder kan fjernes fra berigede populationer ved FACS-sortering. For at estimere renhed berigede populationer, gate på levende celler baseret på forward og side sc...

Watch this Scientific Journal Video about Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function at JoVE.com

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

En enkel, men effektiv metode, der benytter magnetiske nanopartikler til at opdage og berige antigen-reaktive B-celler til funktionel og fænotypisk analyse beskrives.

Detektion og berigelse af Rare Antigen-specifikke B-Celler til analyse af fænotype og funktion

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

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