Name: assessing somatic hypermutation in ramos b cells after overexpression or knockdown of specific genes
Uploaded: 2011-11-01T13:00:00
Description: Watch this Scientific Journal Video about Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes at JoVE.com

De pMSCV-AID-I-Thy1.1 en pKat2 vectoren werden een soort geschenk van DG Schatz en de pVSV-G, pRSV-Rev, en pMDLg / pRRE vectoren werden een soort geschenk van BR Cullen.

1. Voorbereiding van de monsters en cellen <ol><li> Voer een middelgrote schaal bereiding van DNA (midiprep) van de overexpressie of shRNA-bevattende constructen en de retrovirale vector verpakking pKat2 of de lentivirale vectoren verpakking pVSV-G, pMDLg / pRRE, en pRSV-Rev 25. Gebruik 6 pg DNA per bouwen en een 4 ug van pKat2 (voor retrovirale infecties) of 1,5 ug DNA voor elk van de pVSV-g, pMDLg / pRRE, en pRSV-Rev verpakking vectoren (voor lentivirale infecties). </li><li> Bereid BOSC 23 media. Voor tr...

<ol>
	<li>Di Noia, J. M., Neuberger, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+antibody+somatic+hypermutation.">Molecular mechanisms of antibody somatic hypermutation.</a> Annu. Rev. Biochem. 76, 1-22 (2007).</li><li>Peled, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biochemistry+of+somatic+hypermutation.">The biochemistry of somatic hypermutation.</a> Annu. Rev. Immunol. 26, 481-511 (2008).</li><li>Longerich, S., Basu, U., Alt, F., Storb, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AID+in+somatic+hypermutation+and+class+switch+recombination.">AID in somatic hypermutation and class switch recombination.</a> Curr. Opin. Immunol. 18, 164-174 (2006).</li><li>Bardwell, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+somatic+hypermutation+and+reduced+class-switch+recombination+in+exonuclease+1-mutant+mice.">Altered somatic hypermutation and reduced class-switch recombination in exonuclease 1-mutant mice.</a> Nat. Immunol. 5, 224-229 (2004).</li><li>Martomo, S. A., Yang, W. W., Gearhart, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+Msh6+but+not+Msh3+in+somatic+hypermutation+and+class+switch+recombination.">A role for Msh6 but not Msh3 in somatic hypermutation and class switch recombination.</a> J. Exp. Med. 200, 61-68 (2004).</li><li>Phung, Q. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+hypermutation+at+G+and+C+nucleotides+in+immunoglobulin+variable+genes+from+mice+deficient+in+the+MSH2+mismatch+repair+protein.">Increased hypermutation at G and C nucleotides in immunoglobulin variable genes from mice deficient in the MSH2 mismatch repair protein.</a> J. Exp. Med. 187, 1745-1751 (1998).</li><li>Rada, C., Ehrenstein, M. R., Neuberger, M. S., Milstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hot+spot+focusing+of+somatic+hypermutation+in+MSH2-deficient+mice+suggests+two+stages+of+mutational+targeting.">Hot spot focusing of somatic hypermutation in MSH2-deficient mice suggests two stages of mutational targeting.</a> Immunity. 9, 135-141 (1998).</li><li>Rada, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoglobulin+isotype+switching+is+inhibited+and+somatic+hypermutation+perturbed+in+UNG-deficient+mice.">Immunoglobulin isotype switching is inhibited and somatic hypermutation perturbed in UNG-deficient mice.</a> Curr. Biol. 12, 1748-1755 (2002).</li><li>Delbos, F., Aoufouchi, S., Faili, A., Weill, J. C., Reynaud, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+polymerase+eta+is+the+sole+contributor+of+A/T+modifications+during+immunoglobulin+gene+hypermutation+in+the+mouse.">DNA polymerase eta is the sole contributor of A/T modifications during immunoglobulin gene hypermutation in the mouse.</a> J. Exp. Med. 204, 17-23 (2007).</li><li>Masuda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+polymerase+eta+is+a+limiting+factor+for+A:T+mutations+in+Ig+genes+and+contributes+to+antibody+affinity+maturation.">DNA polymerase eta is a limiting factor for A:T mutations in Ig genes and contributes to antibody affinity maturation.</a> Eur. J. Immunol. 38, 2796-2805 (2008).</li><li>Lim, D. S., Hasty, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutation+in+mouse+rad51+results+in+an+early+embryonic+lethal+that+is+suppressed+by+a+mutation+in+p53.">A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53.</a> Mol. Cell. Biol. 16, 7133-7143 (1996).</li><li>Xiao, Y., Weaver, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+gene+targeted+deletion+by+Cre+recombinase+demonstrates+the+requirement+for+the+double-strand+break+repair+Mre11+protein+in+murine+embryonic+stem+cells.">Conditional gene targeted deletion by Cre recombinase demonstrates the requirement for the double-strand break repair Mre11 protein in murine embryonic stem cells.</a> Nucleic. Acids. Res. 25, 2985-2991 (1997).</li><li>Zhu, J., Petersen, S., Tessarollo, L., Nussenzweig, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+the+Nijmegen+breakage+syndrome+gene+NBS1+leads+to+early+embryonic+lethality+in+mice.">Targeted disruption of the Nijmegen breakage syndrome gene NBS1 leads to early embryonic lethality in mice.</a> Curr. Biol. 11, 105-109 (2001).</li><li>Luo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+mRad50+causes+embryonic+stem+cell+lethality,+abnormal+embryonic+development,+and+sensitivity+to+ionizing+radiation.">Disruption of mRad50 causes embryonic stem cell lethality, abnormal embryonic development, and sensitivity to ionizing radiation.</a> Proc. Natl. Acad. Sci. U. S. A. 96, 7376-7381 (1999).</li><li>Pavri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation-induced+cytidine+deaminase+targets+DNA+at+sites+of+RNA+polymerase+II+stalling+by+interaction+with+Spt5.">Activation-induced cytidine deaminase targets DNA at sites of RNA polymerase II stalling by interaction with Spt5.</a> Cell. 143, 122-133 (2010).</li><li>Lee-Theilen, M., Matthews, A. J., Kelly, D., Zheng, S., Chaudhuri, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CtIP+promotes+microhomology-mediated+alternative+end+joining+during+class-switch+recombination.">CtIP promotes microhomology-mediated alternative end joining during class-switch recombination.</a> Nat. Struct. Mol. Biol. 18, 75-79 (2011).</li><li>Basu, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA+exosome+targets+the+AID+cytidine+deaminase+to+both+strands+of+transcribed+duplex+DNA+substrates.">The RNA exosome targets the AID cytidine deaminase to both strands of transcribed duplex DNA substrates.</a> Cell. 144, 353-363 (2011).</li><li>Yabuki, M., Fujii, M. M., Maizels, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MRE11-RAD50-NBS1+complex+accelerates+somatic+hypermutation+and+gene+conversion+of+immunoglobulin+variable+regions.">The MRE11-RAD50-NBS1 complex accelerates somatic hypermutation and gene conversion of immunoglobulin variable regions.</a> Nat. Immunol. 6, 730-736 (2005).</li><li>Sale, J. E., Neuberger, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TdT-accessible+breaks+are+scattered+over+the+immunoglobulin+V+domain+in+a+constitutively+hypermutating+B+cell+line.">TdT-accessible breaks are scattered over the immunoglobulin V domain in a constitutively hypermutating B cell line.</a> Immunity. 9, 859-869 (1998).</li><li>Cumbers, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+iterative+affinity+maturation+of+antibodies+in+vitro+using+hypermutating+B-cell+lines.">Generation and iterative affinity maturation of antibodies in vitro using hypermutating B-cell lines.</a> Nat. Biotechnol. 20, 1129-1134 (2002).</li><li>Papavasiliou, F. N., Schatz, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cycle-regulated+DNA+double-stranded+breaks+in+somatic+hypermutation+of+immunoglobulin+genes.">Cell-cycle-regulated DNA double-stranded breaks in somatic hypermutation of immunoglobulin genes.</a> Nature. 408, 216-221 (2000).</li><li>Parsa, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AID+mutates+a+non-immunoglobulin+transgene+independent+of+chromosomal+position.">AID mutates a non-immunoglobulin transgene independent of chromosomal position.</a> Mol. Immunol. 44, 567-575 (2007).</li><li>Zhang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+instability+of+V+region+hypermutation+in+the+Ramos+Burkitt's+lymphoma+cell+line.">Clonal instability of V region hypermutation in the Ramos Burkitt's lymphoma cell line.</a> Int. Immunol. 13, 1175-1184 (2001).</li><li>Ukai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+a:T+mutations+is+dependent+on+cellular+environment+but+independent+of+mutation+frequency+and+target+gene+location.">Induction of a:T mutations is dependent on cellular environment but independent of mutation frequency and target gene location.</a> J. Immunol. 181, 7835-7842 (2008).</li><li>Dull, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+third-generation+lentivirus+vector+with+a+conditional+packaging+system.">A third-generation lentivirus vector with a conditional packaging system.</a> J. Virol. 72, 8463-8471 (1998).</li><li>Nakamura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+frequency+class+switching+of+an+IgM++B+lymphoma+clone+CH12F3+to+IgA++cells.">High frequency class switching of an IgM+ B lymphoma clone CH12F3 to IgA+ cells.</a> Int. Immunol. 8, 193-201 (1996).</li><li>Muramatsu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+expression+of+activation-induced+cytidine+deaminase+(AID),+a+novel+member+of+the+RNA-editing+deaminase+family+in+germinal+center+B+cells.">Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells.</a> J. Biol. Chem. 274, 18470-18476 (1999).</li></ol>

Zoals eerder besproken, hebben cellijn modellen voor antilichaam diversificatie uitgegroeid tot een populaire uitvalsbasis om nieuwe eiwitten die verschillende stappen invloed tijdens antilichaam diversificatie te identificeren. We presenteren hier een methode voor het gebruik van virale infectie van beide knock-down of overexpressie eiwitten in de Ramos B-cellijn en vervolgens onderzoekt de impact op de SHM. Voor deze studies maken we gebruik van zowel WT Ramos en AID hi Ramos ce...

Aanbevolen reagentia - de meeste van deze kan worden vervangen door soortgelijke producten van andere leveranciers. <table border="1" ><tbody><tr><th> Naam van het reagens </th><th > Vennootschap </th><th> Catalogus nummer </th><th> Reacties </th></tr><tr><td > 6-well duidelijke TC-behandelde platen </td><td > Corning </td><td> 3516 </td><td ></td></tr><tr><td > 10 ml BD Luer-Loksyringes </td><td > BD Medical </td><td > 309604 </td><td ></td></tr><tr><td> 24-well duidelijke TC-behandelde platen </td><td > Corning </td><td > 3526 </td><td></td></tr><tr><td > 96-well duidelijke vlakke bodem polystyreen TC-behandeld microplaten </td><td > Corning </td><td > 3596 </td><td ></td></tr><tr><td > 100 mm TC-behandeld kweekschalen </td><td > Corning </td><td > 430167 </td><td ></td></tr><tr><td > Acrodisc spuit filters, 0,45 pm </td><td > Pall Life Sciences </td><td > 4604 </td><td ></td></tr><tr><td > Agar-agar </td><td > Teknova </td><td > A7777 </td&gt; <td ></td></tr><tr><td > Agarose </td><td > GeneMate </td><td> E-3120-500 </td><td ></td></tr><tr><td > Ampicilline </td><td> Sigma </td><td > A0166 </td><td> 100 mg / ml in water </td></tr><tr><td > BD FACSCanto II flowcytometer </td><td> BD Biosciences </td><td ></td><td > of soortgelijke </td></tr><tr><td> BD Falcon ronde bodem polystyreen buizen </td><td> BD Biosciences </td><td > 352054 </td><td > voor FACS </td></tr><tr><td > BOSC 23 cellen </td><td > ATCC </td><td > CRL-11270 </td><td ></td></tr><tr><td > CO 2 incubator die in staat van 37 ° C </td><td ></td><td ></td><td ></td></tr><tr><td > DMEM (Dulbecco&#39;s gemodificeerd Eagle&#39;s medium) </td><td > Sigma </td><td> D6429 </td><td ></td></tr><tr><td > FBS (foetaal runderserum) </td><td > Gemini Bio-producten </td><td > 100-106 </td><td ></td></tr><tr><td > FITC α-rat CD90/mouse CD90.1 antilichaam </td><td > BioLegend </td><td > 202503 </td><td > FITCα-Thy1.1 </td></tr><tr><td > Fugene 6 transfectiereagens </td><td > Roche </td><td > 11814443001 </td><td></td></tr><tr><td > HEPES bufferoplossing </td><td > Invitrogen </td><td > 15630-080 </td><td ></td></tr><tr><td > KAPA HiFi DNA-polymerase </td><td > KAPA Biosystems </td><td > KK2101 </td><td ></td></tr><tr><td > LB bouillon (lysogeny bouillon - Luria) Poeder </td><td > Difco </td><td > 240230 </td><td ></td></tr><tr><td > MISSIE TRC shRNA bacteriële glycerol voorraad </td><td > Sigma </td><td ></td><td> shRNA vectoren </td></tr><tr><td > NCS (pasgeboren kalf serum) </td><td > Gemini Bio-producten </td><td > 100-504 </td><td ></td></tr><tr><td> PBS (fosfaat gebufferde oplossing) </td><td > Invitrogen </td><td> 70011 </td><td > verdund tot 1x in het water </td></tr><tr><td > PE α-humaan IgM antilichaam </td><td > BioLegend </td><td > 314508 </td><td ></td></tr><tr><td > PGS (penicilline-streptomycine-glutamine oplossing) </td><td > Gemini Bio-Producten </td><td > 400-110 </td><td ></td></tr><tr><td> Polybreen (hexadimethrine bromide) </td><td > Sigma </td><td > 107689 </td><td > 10 mg / ml in water </td></tr><tr><td > PureYield Plasmide Midiprep System </td><td > Promega </td><td > A2495 </td><td></td></tr><tr><td> Puromycine </td><td> Sigma </td><td > P8833 </td><td > 250 pg / ml in water </td></tr><tr><td > QIAquick gelextractiekit </td><td > QIAGEN </td><td > 28706 </td><td ></td></tr><tr><td> Ramos (RA 1) cellen </td><td > ATCC </td><td > CRL-1596 </td><td ></td></tr><tr><td > RPMI-1640 medium </td><td > Sigma </td><td > R8758 </td><td ></td></tr><tr><td > SuperScript II </td><td > Invitrogen </td><td > 18064-022 </td><td></td></tr><tr><td > SYBR FAST qPCR kit </td><td > KAPA Biosystems </td><td > KK4601 </td><td ></td></tr><tr><td > Taq DNA-polymerase </td><td > Invitrogen </td><td > 18038-042 </td><td ></td></tr><tr><td > TOPO TA Cloning Kit </td><td > Vitrogen </td><td > K4520-01 </td><td ></td></tr><tr><td > TRIzol </td><td > Invitrogen </td><td > 15596-026 </td><td ></td></tr><tr><td > Wizard SV Genomic DNA-zuiveringssysteem </td><td > Promega </td><td > A2361 </td><td ></td></tr><tr><td > X-Gal [5-broom-4-chloor-3-indoyl-β-D-galatopyranoside] </td><td > Growcells </td><td > C-5687 </td><td > 40 mg / ml in DMSO </td></tr></tbody></table>

assessing somatic hypermutation in ramos b cells after overexpression or knockdown of specific genes

B-cellen beginnen hun leven met een lage affiniteit antilichamen gegenereerd door V (D) J recombinatie. Echter, bij het ​​opsporen van een ziekteverwekker, is de variabele (V) regio van een immunoglobuline (Ig) gen ongeveer 100.000 keer meer dan de rest van het genoom door middel van somatische hypermutatie (SHM) gemuteerd, wat resulteert in een hoge affiniteit antilichamen 1,2. Daarnaast, klasse switch recombinatie (CSR) produceert antilichamen met verschillende effector functies, afhankelijk van de aard van de immuunrespons die nodig is voor een bepaalde ziekteverwekker. CSR en SHM worden geïnitieerd door de activatie-geïnduceerde cytidinedeaminase (AID), die cytosine residuen in DNA te produceren uracils deaminates. Deze uracils worden verwerkt door foutgevoelige vormen van reparatie wegen, uiteindelijk leidend tot mutaties en recombinatie 1-3. Onze huidige kennis van de moleculaire details van de SHM en MVO komen uit een combinatie van studies in muizen, primaire cellen, cellijnen, en cel-free experimenten. Muismodellen blijft de gouden standaard met genetische knockouts tonen cruciale rol voor vele reparatie factoren (bijvoorbeeld Ung, MSH2, MSH6, Exo1, en ​​polymerase η) 4-10. Echter, niet alle genen zijn vatbaar voor de knock-out studies. Bijvoorbeeld, knockouts van verschillende double-strand break repair eiwitten embryonaal dodelijk of aantasting B-cel ontwikkeling 11-14. Bovendien, soms de specifieke functie van een eiwit in SHM of MVO kan worden gemaskeerd door de meer globale defecten veroorzaakt door de knock-out. Daarnaast kan worden omdat experimenten in muizen langdurig, veranderen van de expressie van individuele genen in cellijnen is uitgegroeid tot een steeds populairder eerste stap naar het identificeren en karakteriseren kandidaat-genen 15-18. Ramos - een Burkitt lymfoom cellijn die constitutief ondergaat SHM - is een populaire cellijn model om SHM 18-24 te bestuderen. Een voordeel van Ramos cellen is dat ze een ingebouwde handige semi hebben-Kwantitatieve meting van SHM. Wild-type cellen brengen IgM en, als ze pick-up mutaties, een aantal van de mutaties knock-out IgM expressie. Daarom is het testen van IgM verlies door fluorescentie-geactiveerde cel scanning (FACS) biedt een snelle uitlezing voor het niveau van de SHM. Een meer kwantitatieve meting van de SHM kan worden verkregen door direct sequencing het antilichaam genen. Omdat Ramos-cellen zijn moeilijk te transfecteren, produceren we stabiele derivaten die zijn verhoogd of verlaagd expressie van een individueel gen door het infecteren van cellen met retrovirale of lentivirale constructen die ofwel een overexpressie cassette of een short hairpin RNA (shRNA), respectievelijk bevatten. Hier beschrijven we hoe we dan Ramos cellen te infecteren en het gebruik van deze cellen om de rol van specifieke genen op de SHM (figuur 1) te onderzoeken.

Watch this Scientific Journal Video about Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes at JoVE.com

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

We beschrijven hoe retrovirale of lentivirale infecties van overexpressie of shRNA-bevattende constructies in het menselijk Ramos B-cellijn en hoe somatische hypermutatie te meten in deze cellen. Uit te voeren

Het beoordelen van somatische hypermutatie in Ramos B-cellen na Overexpressie of Knockdown van specifieke genen

B cells start their life with low affinity antibodies generated by V(D)J recombination.  However, upon detecting a pathogen, the variable (V) region of an ...

Research

JoVE Journal

Immunology and Infection

Murine Model of CD40-activation of B cells

Muizenmodel van CD40-activatie van B-cellen

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

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

Inductie en beoordeling van klasse Switch recombinatie in gezuiverd Murine B-cellen

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 ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Genereren van Multivirus-specifieke T-cellen te voorkomen / virale infecties te behandelen na allogene hematopoietische stamceltransplantatie

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Following Cell-fate in E. coli After Infection by Phage Lambda

Following Cell-fate in E. coli After Infection by Phage Lambda

Na Cell-lot in<em&gt; E. coli</em&gt; Na Infectie door Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Detection of True IgE-expressing Mouse B Lineage Cells

Detectie van True IgE-expressie muis B Lineage Cellen

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

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

Detectie en Verrijking van zeldzame antigeen-specifieke B-cellen voor analyse van fenotype en functie

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 ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpressie en zuivering van het menselijk<em&gt; Cis</em&gt; -prenyltransferase in<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Generatie van discriminatoire menselijke monoklonale antistoffen uit zeldzame antigeen-specifieke B-cellen in bloed circuleert

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding

Beoordeling van cellulaire Stress en ontsteking in Discrete oxytocine-afscheidende kernen van de hersenen in de neonatale Rat vóór en na de eerste biest voeding

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression ...

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

Analyse van Shear Flow-geïnduceerde migratie van RattenUitrustingen Marginal Zone B cellen 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, ...