FormalinFixedCellPlates

TrypsinizeconfluentflasksPoolandcountcellsCentrifugeat1500rpmfor10minutesResUSPendtotheappropriateconcentrationincompletemedium4x105cells/mlforepithelialcells2x105cells/mlforfibroblastcellsAdd100ml/cellto96wellcultureplates.Incubateovernightat37oC.WashplatestwicewithPBSAdd125ml/well10%BufferedFormalinFixfor15minutesatroomtemperatureWashthreetimeswithdi-H2O.Blotdry.Storeat2-8oC.Reagents

PBS:1%BSAPBS:2%BSACarbonateBuffer1.59gNa2CO32.93gNaHCO3Dissolvein900mldi-H2O.CheckpHandadjustto9.6necessary.Qs.to1liter.10XSubstrateBuffer,pH6.036.6gCitricAcid,monohydrate113.5gPotassiumdibasicphosphateDissolvein900mldi-H2O.CheckpHandadjustto6.0ifnecessary.Qs.to1liter.0.3%H2O2Dilute30%stockPeroxide1:100indi-H2O.OPDStock,4.0%4gOPDin100mldi-H2O.Aliquotandstoreat-20oC.Protectfromlight.4.5NH2SO412.0mlConcentratedSulfuricAcid88.0mldi-H2OProcedure

WashELISAplatesoncewithdi-H2O.Add250ml/wellPBS:2%BSA.Incubate1hourat37oC.Wash3timeswithdi-H2O.Add50ml/wellsupe,ascites,orcontrolsdilutedinPBS:1%BSA.Incubatefor2hrat37oC.Wash5timeswithdi-H2O.Add50ml/wellanti-mouseIgG:HRPdilutedinPBS:1%BSA.Incubatefor1hrat37oC.Wash5timeswithdi-H2O.Washoncewithcarbonatebuffer.Add50ml/wellworkingsubstratesolution0.5ml4.0%OPD5ml30%H2O21.0ml10XSubstratebuffer8.5mldi-H2O.Incubatefor20minutesatroomtemperature.Add25ml/well4.5NSulfuricAcidReadA490Notes

Testallsupernatantsat1:5dilution.Testascitesat1:100

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