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Monoclonal Antibodies
蚂蚁淘
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Iftitreispositivedooneofthefollowing2weekslater:
- Boosttailveinwith20ug-50ugofAntigeninPBSandproceedwithfusiononday4.OR
- Boostsubcutaneouslywith50ug-100ugofAntigeninPBSandproceedwithfusion4dayslater.OR
- Boost3daysinarowwith15ugAginPBSandproceedwithfusionon4thday.
CellLines:
- Myeloma;P3X63-Ag8.653
- Origin:BALB/c,nonsecreting,8-azaguanineresistant,HPRT-. - Myelomafox-NY
- Origin:Robertsonian,8-azaguanineresistant,HPRT-,APRT-. - (micehaveresistancetodrugandexpressionofheavychainonthesamechromosome). - Macrophage-derivedJ774A.1
Maintenanceofthecells:
Stocksolutions:
| - | IMDM: |
| - | FetalCalfSerum- |
| - | Transferring:Ironsaturated.1000Xstock=1mg/ml |
| - | HTsupps:50XfromSigmaH0137(Storeat-20°C). |
| - | 2-Mercaptoethanol:1000Xstock(5x10-2).(Storeat4°C.) |
| - | ATsupplement:50Xstock,SigmaA-7422.(Storeat-20°C.) |
| - | KanamycinSulfate:100XfromGibco-BRL600-5160AG.(Storealiquotsat-20°C) |
| - | MCM:MacrophageConditionedMedium.(usedinsteadoffeedercells) |
Seedmacrophagesatadensityof1.5x105cells/mlinthemediumdescribedonnextpage.Add2.5ug/mlLPSwhichinducesdifferentiation.
Collectsupafter2-3days,orwhenmediumisgettingtooyellow.Induce2moretimes,eachtimewith1ug/mlLPSandcollectsupafter2dayseach.
Poolthesups,filteranduseasrecommended.(Couldbealiquotedandstoredat-20C.)
PreparationofMedia:
forP3X63-Ag8.653:IMDMcompleteto425mlofIMDMadd:
| - | 0.5ml1000Xtransferring |
| - | 0.5ml1000X2-Mercaptoethanol |
| - | 10ml50XHT |
| - | 5ml100XKanamycinSulfate |
| - | 75mlFCS(final15) |
forfox-NY:IMDMorRPMI
| - | 10FCS |
| - | 1XATsupplement |
| - | transferring |
| - | Kanamycin |
forJ774A.1:samemediumastheonefortheP3X63-Ag8.653(=Ag8).
Growthconditions:
| - | Allcelllinesmentionedabovegrowat37°C,7CO2. |
| - | TheMyelomasoptimaldensityis3.5x105/ml. |
TheMacrophagesoptimaldensityis1.5x105/ml.Whenexpandingthem,usea"policeman"toscrapethem;thisiseasiertodoiftheyaregrowinginpetridishesatthisstage.
FreezingHybridoma/Myeloma/macrophages.
Freezingsolution:90FCS+10DMSO,icecold.
- Spindown107cells(106minimum)at1200rpmfor5min.
- Aspiratemedium.
- ResUSPendin1mloficecoldfreezingsolution.
- Transfervialtoaninsulatedfreezingboxandplaceat-70°Cforatleast1hr.(couldbeforacoupleofdays).
- Transferthevialfromthe-70°Ctotheliquidnitrogentankandlogtheentryinthefreezerlog.
Thawingcells:
- Takevialoutofliquidnitrogentankandthawitimmediatelyina37°Cbath(about1min).
- Whenthereisstillasmallpieceoficeleft,dilutethecellsbytransferringthemintoaconicaltubecontaining10mlofthegrowthmediumat37°C.
- Spinat1200rpmfor5min.
- Aspiratemediumandresuspendcellsin5mlofmedium,ina25cm2flask.
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