Name: detection of true ige-expressing mouse b lineage cells
Uploaded: 2014-12-01T14:00:00
Description: Watch this Scientific Journal Video about Detection of True IgE-expressing Mouse B Lineage Cells at JoVE.com

DRW støttes av NIH tilskudd AI089972 og AI113217, og ved Mucosal Immunologi Studier Team, og har en karriere Award for Medisinsk Forskere fra Burroughs Wellcome Fondet.

MERK: Alle forsøkene beskrevet her var i samsvar med The Institutional Animal Care og bruk Committee (IACUC) retningslinjer og godkjent av Forsøksdyrbarnesykehus (ARCH), Boston, Mass. 1. Utarbeidelse av reagenser <ol><li> 1,000x IL-4 Lager: Fortynn cytokin IL-4 til 20 pg / ml i H2O eller 0,1% bovint serumalbumin (BSA). Dispensere i 200 ul porsjoner til 1,5 ml mikrosentrifugerør og butikk ved -20 ° C. </li><li> 1,000x anti-CD40: Skaff fra produsenten på 1,0 mg / ml, oppbevares ved 4 ° C. </li><...

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Stimulering av mus splenic B-celler i kultur med anti-CD40 og IL-4 vil simulere T helper type 2 (T H 2) interaksjoner, oppmuntrende klasse veksling til IgG1 og IgE 5. B-celler kan aktiveres for CSR i sammenheng med totale splenocytes 12 eller som rene milt B-celler 11. Som nevnt i protokollen (trinn 2.3), er B-celle berikelse valgfritt, og er på skjønn av eksperimentator å fastslå om det ville gunstig. Positive magnetisk separasjon av B-celler ved hjelp av CD45R (B220) -me...

Under immunoglobulin tung kjede (avIgH) klasse bryter rekombinasjon (CSR) i mus og mennesker, μ Igh konstant område eksoner (Cμ) slettes og erstattes med en av flere sett med nedstrøms konstant område eksoner (C H r) (f.eks Cγ, Cε, og Cα), noe som resulterer i en bryter fra produksjonen av IgM til produksjon av andre Ig-klasser (for eksempel IgG, IgE, IgA eller). CSR skjer innen switch (S) regioner, som er 1-10 kb sekvenser lokalisert 5 &#39;til hvert sett med C H s 1. Aktiverings-Induced cytidindeaminase (AID) enzym initierer CSR via cytidine deaminering aktivitet. IgE er en viktig formidler av allergisk sykdom to og en større forståelse av hvordan IgE produseres og regulert kan åpne dører til nye terapeutiske tilnærminger til atopisk sykdom. Hos mus og mennesker, er IgE den mest strengt regulert Ig isotypen. IgE er normalt oppdages på nivåer tusenvis av times mindre enn andre avIgH isotyper tre, men kan være sterkt forhøyet i sykdomstilstander 4. Imidlertid er CSR til IgE ufullstendig forstått. In vitro-aktivering av B-celler ved hjelp av IL-4 i kombinasjon med enten anti-CD40 eller LPS induserer CSR til både lgG1 og IgE 5. Aktiverte B-celler uttrykker den lave affinitet IgE reseptor FcεRII / CD23 2,6, som binder løselig IgE utskilles i kultur etter CSR. Derfor når analysere med flowcytometri, B-celler med reseptor-bundet IgE flekken på samme måte som B-celler endogent uttrykker IgE 7. Mens det er kjent at mus B celler uttrykker den lave affinitet IgG reseptor FcγRIIB1 (CD32) 8, i vår erfaring det ser ikke ut til å forstyrre måling av klassen bytte til IgG1. Imidlertid, når man måler IgE veksling etter at B-celleaktivering i kultur, kan ikke-spesifikk overflate-bundet IgE tilsløre analysen. Med vanlige fargemetoder, ikke-IgE-uttrykker celler flekken positivt for IgE. Skissert her er en strategi som har vært benyttet til å gjennomføre samfunnsansvar analyser og oppdage ekte IgE-uttrykke B-celler fra mus 9-11. Behandling av aktiverte B-celler med trypsin, til en felles lab reagens fordøye protein, fjerner både cytophylic og membranbundet overflate IgE. Påfølgende permeabilization og farging for cytoplasmic IgE avslører dermed den sanne IgE-produserende celler.

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	<tbody>
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			<td height="37" style="height:37px;width:301px;">Name</td>
			<td style="width:179px;">Company</td>
			<td style="width:135px;">Catalog Number</td>
			<td style="width:343px;">Comments</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Anti-Mouse IgE FITC</td>
			<td>BD Pharmingen</td>
			<td>553415</td>
			<td>clone R35-72</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Rat Anti-Mouse IgG1 PE</td>
			<td>BD Pharmingen</td>
			<td>550083</td>
			<td>clone A85-1</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Methanol</td>
			<td>BDH</td>
			<td>BDH1135-4LP</td>
			<td>Keep at -20 &#176;C</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Falcon Cell Strainer 40 &#181;m Nylon</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Falcon Cell Strainer 70 &#181;m Nylon</td>
			<td>Corning</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Anti-Hu/Mo CD45R(B220) PerCP-Cy5.5</td>
			<td>eBioscience</td>
			<td>45-4052-82</td>
			<td>clone RA3-6B2</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">anti-Mouse/Rat CD40</td>
			<td>eBioscience</td>
			<td>16-0402-85</td>
			<td>clone HM40-3</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">RPMI Medium 1640</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">HEPES (1M)</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">MEM-NEAA (100X)</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Phosphate Buffered Saline (10X)</td>
			<td>Lifetechnologies (Corning)</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr height="66">
			<td height="66" style="height:67px;">MACS CD45R (B220) microbeads&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-049-501</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">MACS purification column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">IL-4</td>
			<td>PeproTech</td>
			<td>214-14</td>
			<td>Reconstitute in water or 0.1% BSA</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Formalin Solution, Neutral Buffered, 10%</td>
			<td>Sigma Aldrich</td>
			<td>HT501128-4L</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Trypsin-EDTA Solution (10X)</td>
			<td>Sigma Aldrich</td>
			<td>T4174-100mL</td>
			<td>5.0g Trypsin, 2.0g EDTA&#376;4Na per Liter of 0.9% NaCl</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Penicillin-Streptomycin</td>
			<td>Sigma Aldrich</td>
			<td>P0781-100mL</td>
			<td>10,000U Penicillin; 10 mg/mL Streptomycin (100X)</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">L-Glutamine 200mM</td>
			<td>Sigma Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">2-mercaptoethanol (99%)</td>
			<td>Sigma Aldrich</td>
			<td>M6250-100mL</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Red cell lysis buffer</td>
			<td>Sigma Aldrich</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Rat Anti-Mouse IgM RPE/cy7</td>
			<td>SouthernBiotech</td>
			<td>1140-17</td>
			<td>clone 1B4B1</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">HyClone Fetal Calf Serum</td>
			<td>Thermo</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr height="161">
			<td height="161" style="height:161px;">B cell Stimulation media</td>
			<td></td>
			<td></td>
			<td style="width:343px;">B cell stimulation media consists of RPMI with fetal calf serum (15%), 20 mM HEPES, 1X MEM-NEAA, 2.0mM L-glutamine,&#160; 1X Penicillin-Streptomycin (Penicillin:100 U/ml, Streptomycin: 100 &#181;g/ml), Beta-mercaptoethanol (7 &#181;L/L), IL-4 (20 ng/mL), and anti-CD40 (1 &#181;g/mL)</td>
		</tr>
	</tbody>
</table>

detection of true ige-expressing mouse b lineage cells

B-lymfocytt-immunoglobulin-tung kjede (avIgH) klasse bryter rekombinasjon (CSR) er en fremgangsmåte hvor utgangspunktet uttrykt IgM bytter til andre igh isotyper, så som IgA, IgE og IgG. Måling av in vitro avIgH CSR er en viktig metode for studiet av en rekke biologiske prosesser som strekker seg fra DNA-rekombinasjon og reparasjon for aspekter av molekylær og cellulær immunologi. In vitro CSR-analysen involverer strømningscytometrisk måling overflate Ig-ekspresjon på aktiverte B-celler. Selv om måling av IgA og IgG subklasser er enkel, er måling av IgE ved denne metode problematisk på grunn av oppløselig IgE-binding til FcεRII / CD23 uttrykkes på overflaten av aktiverte B-celler. Her beskriver vi en unik fremgangsmåte for nøyaktig måling av IgE-produserende mus-B-celler som har gjennomgått CSR i kultur. Metoden er basert på trypsin-formidlet kløyving av IgE-CD23 komplekser på celleoverflater, noe som åpner for deteksjon av IgE-produserende B-celler ved avstamning cytoplasmic farging. Denne prosedyren gir en praktisk løsning for flowcytometrisk analyse av CSR til IgE.

Denne prosedyren har blitt implementert for å studere CSR til IgE i mus B-celler. For å demonstrere effektiviteten ved måling av CSR, vi stimulert muse milt-B-celler med anti-CD40 og IL-4 som beskrevet tidligere 11. Etter fem dager etter stimulering ble cellene innsamlet og prosessert ved å bruke den protokoll som er beskrevet ovenfor, og farget med fluorescens-merket IgM, IgG1, IgE, og B220 (CD45R) antistoffer. Lukket på sprengnings lymfocytter (figur 1), en klar populasjon av IgE + cel...

Watch this Scientific Journal Video about Detection of True IgE-expressing Mouse B Lineage Cells at JoVE.com

Detection of True IgE-expressing Mouse B Lineage Cells

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

Påvisning av True IgE-uttrykke Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

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