Name: genome-wide analysis of hdac inhibitor-mediated modulation of micrornas and mrnas in b cells induced to undergo class-switch dna recombination and plasma cell differentiation
Uploaded: 2017-09-20T12:30:00
Description: Watch this Scientific Journal Video about Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell

Detta arbete fick stöd av NIH grants AI 105813 och AI 079705 (till PC), bevilja alliansen för Lupus forskning Target identifiering i Lupus Grant ALR 295955 (till PC) och artrit National Research Foundation forskningen (HZ). TS stöddes av Pediatrics Medical Center, andra Xiangya sjukhuset, Central South University, Changsha, Kina, i samband med Xiangya-UT skolan läkemedel San Antonio medicinsk student besöker programmet.

protokollet följer djurvård riktlinjer The institutionsvård av djur och användning kommittéer vid University of Texas Health Science Center på San Antonio.
 1. stimulering av mus B celler för CSR, Plasma celldifferentiering och HDI behandling <ol><li>beredning av mjälte cellsuspensioner Obs: utföra alla steg i en LAF förutom mus dödshjälp och dissektion.
	<ol><li>Euthanize den specifikt patogenfria C57BL/6J mus (8-12 veckors ålder) av CO 2 inan...

<ol>
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J., Mai, T., Casali, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoglobulin+class-switch+DNA+recombination:+induction,+targeting+and+beyond.">Immunoglobulin class-switch DNA recombination: induction, targeting and beyond.</a> Nat. Rev. Immunol. 12, 517-531 (2012).</li><li>Nutt, S. L., Hodgkin, P. D., Tarlinton, D. M., Corcoran, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+generation+of+antibody-secreting+plasma+cells.">The generation of antibody-secreting plasma cells.</a> Nat. Rev. Immunol. 15, 160-171 (2015).</li><li>Bartel, D. 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A., 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=Histone+deacetylase+inhibitors+upregulate+B+cell+microRNAs+that+silence+AID+and+Blimp-1+expression+for+epigenetic+modulation+of+antibody+and+autoantibody+responses.">Histone deacetylase inhibitors upregulate B cell microRNAs that silence AID and Blimp-1 expression for epigenetic modulation of antibody and autoantibody responses.</a> J Immunol. 193, 5933-5950 (2014).</li><li>Farh, K. K., 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=Genetic+and+epigenetic+fine+mapping+of+causal+autoimmune+disease+variants.">Genetic and epigenetic fine mapping of causal autoimmune disease variants.</a> Nature. 518, 337-343 (2015).</li><li>Nagalakshmi, U., Waern, K., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+method+for+comprehensive+transcriptome+analysis.">RNA-Seq: a method for comprehensive transcriptome analysis.</a> Curr Protoc Mol Biol. , 11-13 (2010).</li><li>Martin, J. 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Immunol. 6, 627 (2015).</li><li>Trapnell, 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=Differential+gene+and+transcript+expression+analysis+of+RNA-seq+experiments+with+TopHat+and+Cufflinks.">Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks.</a> Nat Protoc. 7, 562-578 (2012).</li><li>Kin, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fRNAdb:+a+platform+for+mining/annotating+functional+RNA+candidates+from+non-coding+RNA+sequences.">fRNAdb: a platform for mining/annotating functional RNA candidates from non-coding RNA sequences.</a> Nucleic Acids Res. 35, D145-D148 (2007).</li><li>Agarwal, V., Bell, G. W., Nam, J. W., Bartel, D. 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Det här protokollet ger omfattande strategier för att framkalla B cell klass växling och plasma celldifferentiering; att analysera deras inverkan av epigenetiska modulatorer, nämligen HDI; och för att påvisa effekten av HDI på mRNA och miRNA uttryck i dessa celler. De flesta av dessa metoder kan också användas för att analysera effekterna av epigenetiska faktor på mänskliga B-cells funktion och mRNA/miRNA uttryck. De qRT-PCR och mRNA-seq/miRNA-seq metoder kan också användas för att analysera B-celler som i...

Epigenetiska märken eller faktorer, såsom DNA-metylering, Histon posttranslationell ändringar och icke-kodande RNAs (inklusive mikroRNA), modulera cellens funktion genom att ändra gen uttryck1. Epigenetiska förändringar reglera B-lymfocyter funktion, till exempel immunglobulin klass-switch DNA-rekombination (CSR), somatisk hypermutation (SHM) och differentiering till B-minnesceller eller plasmaceller, därigenom modulera den antikropp och autoantikroppar svar2,3. CSR och SHM kräver kritiskt aktivering-inducerad cytidindeaminas (stöd, kodad som Aicda), som mycket framkallas i B-celler som svar på T-beroende och T-oberoende antigen4. Klass-bytte/hypermutated B-celler ytterligare differentieras till plasmaceller, som utsöndrar stora mängder antikroppar i ett mode som är kritiskt beroende av B-lymfocyter-inducerad mognad protein 1 (Blimp1, Prdm1-kodad)5. Onormala epigenetiska förändringar i B-celler kan leda till avvikande antikropp/autoantikroppar svar, vilket kan leda till allergisk reaktion eller autoimmunitet1,4. Förstå hur epigenetiska faktorer, såsom MicroRNA, modulera B cell-inneboende genuttryck är inte bara viktigt för utvecklingen av vaccin, men är också viktigt att avslöja mekanismerna av potentiella onormala antikroppar/autoantikroppar svaren.
Histon acetylering och deacetylering är ändringar av lysin rester på Histon proteiner normalt katalyseras av Histon kolinacetyltransferas (HAT) och Histon histondeacetylas (HDAC). Dessa ändringar leder till ökande eller minskande tillgänglighet av kromatin och ytterligare tillåta eller förhindra bindning av transkriptionsfaktorer eller proteiner till DNA och förändringen av genen uttryck5,6, 7 , 8. HDAC-hämmare (HDI) är en klass av föreningar som stör funktionen av HDAC. Här, brukade vi HDI (VPA) adress regleringen av HDAC på inneboende gen uttryck profilen av B-celler och på dess mekanism.
MicroRNA är små, icke-kodande RNAs cirka 18 till 22 nukleotider i längd som genereras genom flera stadier. miRNA värd gener transkriberas och bilda hårnål primära mikroRNA (pri-MicroRNA). De exporteras till cytoplasman, där pri-MicroRNA vidarebearbetas till föregångare MicroRNA (pre-MicroRNA). Slutligen bildas mogna MicroRNA genom klyvning av den pre-microRNA. MicroRNA igen de kompletterande sekvenserna inom 3' oöversatta regionen av deras mål mRNA6,7. Genom post-transcriptional ljuddämpning, reglera MicroRNA cellulär aktivitet, såsom proliferation, differentiering och apoptos10,11. Eftersom flera MicroRNA kan rikta samma mRNA, och en enda miRNA kan potentiellt rikta flera mRNA, är det viktigt att ha en sammanhangsberoende utsikt över miRNA uttryck profilen att förstå värdet av enskilda och kollektiva effekten av microRNA. MicroRNA har visat sig vara inblandade i B-cells utveckling och perifera differentiering, samt B-cellen scenen-specifika differentiering, antikroppssvar och autoimmunitet1,4,9. I de 3' UTR Aicda och Prdm1finns det flera validerade eller förutsedd evolutionärt bevarade webbplatser som kan riktas av MicroRNA8.
Epigenetiska modulering, inklusive Histon efter transkriptionen modifiering och MicroRNA, Visa ett cell-typ och cellen scenen-specifik förordning mönster av gen uttryck9. Här, beskriver vi metoder för att definiera HDI-medierad moduleringen av miRNA och mRNA uttryck, CSR och plasma celldifferentiering. Dessa inkluderar protokoll för att inducera B-celler genomgår CSR och plasma celldifferentiering; för behandling av B-celler med HDI; och för att analysera miRNA och mRNA uttryck av qRT-PCR, miRNA-seq och mRNA-seq10,11,12,8,13.

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			<td height="21" style="height:21px;width:528px;">C57BL/6 mice</td>
			<td style="width:152px;">Jackson Labs</td>
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			<td>Hyclone</td>
			<td>SH300</td>
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			<td>Fisher Scientific - Hyclone</td>
			<td>SV3007901&#160;</td>
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			<td>Fisher Scientific</td>
			<td>44-420-3250ML</td>
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			<td>Fisher Scientific</td>
			<td>21008-952&#160;&#160;</td>
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			<td>GIBCO/Life Technologies/Inv</td>
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			<td>Fisher Scientific</td>
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			<td>BioLegend&#160;</td>
			<td>142513 (25 ug)</td>
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			<td>BioLegend</td>
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			<td>Sigma Aldrich</td>
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			<td>Fisher Scientific</td>
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			<td>BioLegend</td>
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			<td>Sigma Aldrich</td>
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			<td>The Baker Company</td>
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			<td>Thermo Scientific</td>
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			<td>Fisher Scientific</td>
			<td>15-462-5Q</td>
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			<td height="21" style="height:21px;">5mL Round Bottom Polystyrene Test Tube</td>
			<td>Fisher Scientific</td>
			<td>&#160;14-959-5</td>
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			<td height="21" style="height:21px;">Corning CentriStar 15ml Centrifuge Tubes&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>05-538-59A</td>
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			<td height="21" style="height:21px;">1.7 mL Microtube, clear</td>
			<td>Genesee</td>
			<td>22-282</td>
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			<td>Fisher Scientific</td>
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			<td>Beckman Coulter</td>
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			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">miRNeasy Mini Kit (50)</td>
			<td>Qiagen</td>
			<td align="right">217004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Direct-zol RNA MiniPrep kit</td>
			<td>Zymo Research</td>
			<td>R2050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform (Approx. 0.75% Ethanol as Preservative/Molecular Biology)</td>
			<td>Fisher Scientific</td>
			<td>BP1145-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rnase-Free Dnase set (50)</td>
			<td>QIAGEN</td>
			<td align="right">79254</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoDrop 2000 Spectrophotometers</td>
			<td>Thermo Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superscript III First-strand Synthesis System RT-PCR</td>
			<td>Invitrogen</td>
			<td align="right">175013897</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">iTaq Universal SYBR Green Supermix</td>
			<td>Bio-rad&#160;</td>
			<td>172-5121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisherbrand 96-Well Semi-Skirted PCR Plates, case of 25</td>
			<td>Fisher</td>
			<td style="width:111px;">14-230-244</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microseal &#39;B&#39; Adhesive Seals</td>
			<td>Bio-Rad</td>
			<td>MSB-1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MyiQ Optics Module</td>
			<td>Bio-Rad</td>
			<td>170-9744</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">iCycler Chassis</td>
			<td>Bio-Rad</td>
			<td>170-8701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optical Kit</td>
			<td>Bio-Rad</td>
			<td>170-9752</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD LSR II Flow Cytometry Analyzer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACSDiva software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FlowJo 10</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2100 Bioanalyzer</td>
			<td>Agilent Technologies</td>
			<td>G2943CA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">S200 Focused-ultrasonicator</td>
			<td>Covaris</td>
			<td>S200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SPRIworks Fragment Library System I for Illumina</td>
			<td>Beckman Coulter</td>
			<td>A288267</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cBot Cluster Generation Station</td>
			<td>illumina</td>
			<td>SY-312-2001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HiSeq 2000 Genome Sequencer</td>
			<td>Illumina</td>
			<td>SY-401-1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TruSeq RNA Library Prep Kit v2</td>
			<td>Illumina</td>
			<td>RS-122-2001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TruSeq Small RNA Library Prep Kit</td>
			<td>Illumina</td>
			<td>RS-200-0012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NEXTflex Illumina Small RNA Sequencing Kit v3</td>
			<td>Bioo Scientific</td>
			<td>5132-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2200 TapeStation</td>
			<td>Agilent</td>
			<td>G2964AA</td>
			<td></td>
		</tr>
	</tbody>
</table>

genome-wide analysis of hdac inhibitor-mediated modulation of micrornas and mrnas in b cells induced to undergo class-switch dna recombination and plasma cell differentiation

Antikroppssvar är fulländade genom flera kritiska B cell-inneboende processer, inklusive somatisk hypermutation (SHM), klass-switch DNA-rekombination (CSR) och plasma celldifferentiering. Under de senaste åren har epigenetiska modifieringar eller faktorer, såsom Histon deacetylering och mikroRNA (Mirna), visats interagera med B-cells genetiska program till formen antikroppssvar, medan dysfunktion av epigenetiska faktorer har visat sig leda till autoantikropp svaren. Analysera genome-wide miRNA och mRNA uttryck i B-celler som svar på epigenetisk modulatorer är viktigt för att förstå epigenetisk reglering av B-cells funktion och antikropp svar. Här visar vi ett protokoll för inducerande B-celler genomgår CSR och plasma cell differentiering, behandling av dessa B-celler med Histon histondeacetylas (HDAC)-hämmare (HDI) och analysera mRNA och mikroRNA uttryck. I detta protokoll, analyserar vi direkt kompletterande DNA (cDNA) sekvenser med nästa generations mRNA sekvensering (mRNA-seq) och miRNA-seq teknik, kartläggning av sekvensering läser till genomet och kvantitativa omvänd Transkription (qRT)-PCR. Med dessa metoder, har vi fastställt att, i B-celler inducerad genomgå CSR och plasma celldifferentiering, HDI, en epigenetisk regulator, selektivt modulerar miRNA och mRNA uttryck och förändrar CSR och plasma cell differentiering.

Med våra protokoll, renat B-celler placeras med LPS (3 µg/mL) och IL-4 (5 ng/mL) för 96 h kan inducera 30-40% av kemikaliesäkerhetsrapporten till IgG1 och ~ 10% av plasma celldifferentiering. Efter behandling med HDI (500µM VPA), kemikaliesäkerhetsrapporten till IgG1 minskat till 10-20%, medan plasma celldifferentiering minskade till ~ 2% (figur 1). HDI-medierad hämning av CSR bekräftades ytterligare minskade antalet efter rekombination Iμ-Cγ1 och mogen VH<...

Watch this Scientific Journal Video about Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell

Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell Differentiation

Epigenetiska faktorer kan interagera med genetiska program att modulera genuttryck och reglera B cellsfunktion. Genom att kombinera in vitro B-cell stimulering, qRT-PCR, och hög genomströmning mikroRNA-sekvens och mRNA-sekvens tillvägagångssätt, kan vi analysera de epigenetiska moduleringen av miRNA och gen uttryck i B-celler.

Genome-wide analys av HDAC-hämmare-medierad modulering av mikroRNA och mRNA i B celler förmås att genomgå klass-switch DNA-rekombination och Plasma celldifferentiering

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA ...

The overall goal of this experiment is to induce B lymphocytes to undergo antibody class-switching and plasma cell differentiation and analyze the modulation as well as epigenetic regulation by the expression of HDAC inhibitor mRNA and microRNA in B-cells. This method can help answer key questions in the B-cell antibody response field such as, epigenetic modulation of immunoglobulin class-switching and plasma cell differentiation. The main advantage of this technique is to provide a simple method to modulate B-cell functions and to define the alteration of microRNAs and mRNAs by epigenetic regulators.After euthanizing a specific pathogen-free C57BL6J mouse and sterilizing the skin according to the text protocol, use autoclaved forceps and dissecting scissors to cut through the skin just below the ribcage to visualize the spleen on the left side of the abdomen, just below the liver. Gently clamp the spleen and cut off all the connecting tissue before extracting the organ. Then place the spleen into a 1.5 milliliter microcentrifuge tube containing 1, 000 microliters of complete RPMI 1640 medium.Next, insert a 70 micron cell strainer into a 50 milliliter polypropylene conical centrifuge tube and pour the spleen from the microcentrifuge tube onto the cell strainer. Use the tip of a 15 milliliter polypropylene conical centrifuge tube to gently mesh the spleen through the strainer. Then with 15 to 20 milliliters of complete medium, rinse the strainer.Spin down the cells at 350 x G for five minutes. Discard the supernatant. Use one milliliter of ACK lysis buffer to re-suspend the cell pellet and incubate for one minute.Then add four milliliters of ACK and incubate the suspension to remove red blood cells from the spleen suspension for an additional four minutes at room temperature with occasional shaking. Then add 25 milliliters of complete medium to quench the lysis reaction. Spin down the cells at 350 x G for five minutes.Discard the supernatant and add one milliliters of complete medium to re-suspend the cell pellet. Then using a hemocytometer, count the viable cells. In a sterile five milliliter polystyrene round bottom tube, prepare a 0.25 to two milliliter cell suspension of 1 x 10 to the 8th cells per milliliter in complete medium.Add normal rat serum to the sample. Next, add 50 microliters per milliliter of isolation cocktail to the tube. Then mix the cells by gently pipetting up and down two to three times and incubate the sample at room temperature for 10 minutes.Add 75 microliters per milliliter of streptavidin encoated magnetic particles to the tube. Gently pipette up and down two to three times and incubate the sample at room temperature for two and a half minutes. Bring the volume up to 2.5 milliliters using complete RPMI 1640 medium and gently pipette two to three times to mix.Place the opened tube into the magnet and incubate it at room temperature for two and a half minutes. Then pour off the supernatant containing untouched B-cells into a new 15 milliliter conical tube. Use trypan blue and a hemocytometer to count the cells.Re-suspend the purified B-cells in complete RPMI 1640 medium at a final concentration of 0.3 x 10 to the 6th cells per milliliter. Then, to the wells of a 48 well plate, add one milliliter of purified B-cells at a 0.3 x 10 to the 6th cells per milliliter three microgram per milliliter of LPS from E.Coli, five nanogram per milliliter of IL4 and either zero or 500 micromolar HDI. Incubate the cells at 37 degree celsius and 5%carbon dioxide for 60 hours for QRT PCR and high throughput mRNA and miRNA sequencing analysis or 96 hours for flow cytometry analysis.After 96 hours of culture, detach the cells from each well by pipetting them up and down several times. Transfer the cell suspension into a 1.5 milliliter microcentrifuge tube. Then, using a bench top centrifuge, spin down the cells at 350 x G for five minutes.Then, discard the supernatant. Mix a cocktail of HBSS buffer and the following reagents, 1%BSA, 0.5 nanograms per milliliter of conjugated goat anti-mouse IgM antibody, 0.2 nanograms per milliliter of conjugated rat anti-mouse IgG1 monoclonal antibody, 0.2 nanograms per milliliter of conjugated rat anti-mouse B220 monoclonal antibody, 0.2 nanograms per milliliter of PE-Cy7-conjugated rat anti-mouse CD138 monoclonal antibody and two nanograms per milliliter of 7-AAD. Incubate the cells in the dark at room temperature for 30 minutes.Then use one milliliter of HBSS with 1%BSA to wash the cells. Use a bench top centrifuge to spin down the cells at 1, 500 x G for five minutes. After discarding the supernatant, use 300 microliters of HBSS with 1%BSA to re-suspend the cells and transfer the cell suspension to a round bottom polystyrene tube.Cover the tube with foil to avoid light exposure. To carry out QRT PCR analysis of mRNA, include the DNAse 1 treatment step. Then, use a commercial total RNA isolation kit that can recover small RNA to extract RNA from 0.2 to 5 x 10 to the 6th cells, following the manufacturer's instructions.After synthesizing cDNA and performing QRT PCR on the mRNA, carry out RT PCR for microRNA using a cyber green real time PCR master mix by thawing at room temperature, 2x PCR master mix, 250 nanomolar miRNA specific forward primer and a universal reverse primer. After preparing a 25 microliter reaction mix according to the text protocol, add 22.5 microliter aliquots per well to a 96 well PCR plate. Then, add 2.5 microliters of template cDNA to the individual wells.Tightly seal the plate film. Then centrifuge the plate at 1, 000 x G at room temperature for one minute. Place the plate in the real time cycler and start the cycling program found in the text protocol.Using the protocol demonstrated in this video, purified B-cells incubated without PS NIL4 for 96 hours can induce 30 to 40%of CSR to IgG1 and approximately 10%of plasma cell differentiation. After treatment with HDI, the CSR to IgG1 decreased to 10 to 20%while plasma cell differentiation decreased to approximately 2%HDI mediated inhibition of CSR was further confirmed by decreased numbers of post-recombination I mu C gamma 1 and mature VHDJHC gamma 1 transcripts. As measured by QRT PCR, the expression of Aicda which is critical for CSR SHM and Prdm1 and Xbp1, which are important for plasma cell differentiation were shown to be inhibited by VPA.As measured by mRNA sequencing of B-cells stimulated without PS Nil4, only 0.3%of these highly expressed mRNAs were upregulated by HDI more than two fold and only 0.36%of the highly expressed mRNAs, including Aicda, Prdm1 and Xbp1 were reduced by HDI more than 50%The miRNA sequencing analysis of miRNA profiling in B-cells treated with HDI or Nil, showed that HDIs selectively upregulate Aicda and Prdm1 targeting miRNAs. Once mastered, this technique can be done in four and half to five hours. While attempting this procedure, it is important to remember to keep a sterile work environment.After watching this video, you should have a good understanding of how to induce B-cells to undergo class-switching and plasma cell differentiation and to modulate these functions by epigenetic regulators.

Research

JoVE Journal

Immunology and Infection

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Induktion och Bedömning av klass Switch rekombination i Renad mus B-celler

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Riktad Differentiering av inducerade pluripotenta stamceller mot T-lymfocyter

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

Inrättande av en<em&gt; In vitro</em&gt; System för att studera intracellulära Beteende<em&gt; Candida glabrata</em&gt; I Human THP-1 makrofager

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Snabb molekylär detektion och differentiering av influensavirus A och B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

Isoleringen, differentiering och kvantifiering av human antikropp-utsöndrande B-celler från blod: ELISpot som en funktionell Avläsning av humoral immunitet

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

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

Detection och anrikning av sällsynta antigenspecifika B-celler för analys av Fenotyp och 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 ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

En In Vivo musmodell åtgärd naiva CD4 T-cellsaktivering, spridning och Th1 differentiering induceras av benmärgen-derived dendritiska celler

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Analys av skjuvning flöde-inducerad Migration av murin marginella zon B-celler In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells

I Vitro differentiering modell av människans normala minne B celler att långlivade plasmaceller

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or ...