Fusionisanimportantproceduretoproducemonoclonalantibodies(MoAb).PROTOCOL- preparethecultureofmyelomacellsandmaintaintheconcentrationofthecellsattheleveloflessthan10^6cells/ml(IMDM+10%FCS/FBS).Justbeforethefusionwash3timesinIMDM(serumfree!),1200rpm,5min,37ѓC.Countcells.
- sacrificethemouseanddissectthespleen.
- teasethespleeninice-coldIMDM(serumfree!);passthroughthenylonfilter;wash3timesinIMDM(serumfree!),1200rpm,5min,37ѓC.Countcells.
- mix10^8spleencells(in25mlofIMDM[serumfree])with2x10^7myelomacells(in25mlofIMDM[serumfree])inFalcon-50mlconicaltube.
- centrifugeat1200rpm,5min,37ѓC.
- aspiratethesupernatant(SN)withaPasteurPipette(alltillthelastdrop!!!)
- gentlybreakthepelletbytappingofthebottomofthetube.
- placethetubeon37ѓCwaterbath;addslowly,dopbydrop1mlofpre-warmed(37ѓC)50%PEG1500(polyethyleneglycol,Roche,Germany)continouslystirringthecellswiththepipettetip(thisprocedurelastsforaperiodover1min)
- add1mlofpre-warmedto37ѓCIMDMcontinouslystirringthemixture(thisprocedurelastsforaperiodover1min).
- add3mlofpre-warmedto37ѓCIMDMcontinouslystirringthemixture(thisprocedurelastsforaperiodover3min).
- add10mlofpre-warmedto37ѓCIMDMcontinouslystirringthemixture(thisprocedurelastsforaperiodover1-2min).
- lettostayfor5minonthe37ѓCwaterbath.
- centrifugeat1200rpm,5min,37ѓC;aspiratetheSN.
- resUSPendthecellsinIMDM-FCS(10%v/v)-HTx100(1%v/v)-AJ(5%v/v)-IL-6,0.5x10^6cells/ml(calculationfiguredoutfromthenumberofspleencellsthefusionwasstartedfrom);lettostayinincubator,37ѓC,pCO27%for2hoursinabigflasc.
- addaminopterin;distributeon96-wellscellcultureplates(Nunc)(200µl/well);placeinincubator(37ѓC,pCO27%)envelopedinaluminiumpaper.
- checkthecultures1-2-7daysafterthefusion.
- onday7-10changetheculturemedium.
- testcloneswhenthayoccupy25%ofthebottomsurfaceofthewell.
SOLUTIONS- IMDM(Iscove"sModifiedDulbecco"sMediumwith25mMHepes/withL-Glutamine)BioWhittakerEuropeCat.:BE12-722F
- IMDM-FCS(10%v/v)-HTx100(1%v/v)-AJ(5%v/v)-IL-6(100U/ml)
- IMDM-FCS(10%v/v)-HTx100(1%v/v)-AJ(5%v/v)-IL6(100U/ml)-aminopterinex100(1%v/v)
- HT(hypoxantine-thymidine)x100,0.22(or0.45)µm-filtered,storeat-20ѓC=hypoxantinesolution,50ml+thymidinesolution,50ml+ddH2Oupto250ml
- hypoxantinesolution=hypoxantine,340mg+ddH2Oupto50ml+NaOH1N,3ml->heatto37ѓC
- thymidinesolution=thymidine(2"-desoxy-thymidine),96mg+ddH2Oupto50ml
- AJ(from"ajouter",toadd,localslang),3L,0.22(or0.45)µm-filtered,storeat-20ѓC=Asp-Argx100,1L+Glutax50,2L+mercaptoethanol,350µl
- Asp-Argx100,1L,pH6.0=L-Asparagine,3.6g(0.24mM)+L-Arginine(0.55mM),11.6g+HCl,0.5N,100ml->heatto37ѓC,addddH2Oupto1L
- Glutax50,1L,pH6.0=glutamine,10.8g(1.48mM)+HCl,0.5N,100ml->heatto37ѓC,addddH2Oupto1L
- aminopterinex100,0.22-0.45µm-filtered,storeat-20ѓC=aminopterine,4.4mg(3.8µM)+ddH2O,50ml+NaOH,1N,100µl->heatto37ѓC,addddH2Oupto250ml
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