Live Cell ELISA Protocol
Live Cell ELISA ProtocolAuthor: Nanci DonackiSource: Contributed by Nanci Donack
| Live Cell ELISA Protocol |
| Author: Nanci Donacki |
| Source: Contributed by Nanci Donacki |
Reagents DMEM:10% FBS DMEM:10% FBS:1% HEPES (DFH) 10X Substrate Buffer, pH 6.0 36.6 g Citric Acid, monohydrate 113.5 g Potassium dibasic phosphate Dissolve in 900 ml di-H2 O. Check pH and adjust to 6.0 if necessary. Qs. to 1 liter. 30% H2 O2 OPD Stock, 4.0% 4 g OPD in 100 ml di-H2 O. Aliquot and store at -20o C. Protect from light. 4.5N H2 SO4 12.0 ml Concentrated Sulfuric Acid 88.0 ml di-H2 O Hanks Balanced Salt Solution (HBSS)Procedure Wash ELISA plates once with 150 ml/well DFH by centrifugation. Add 50 ml/well supe, ascites, or controls diluted in DFH. Incubate for 2 hr at 37o C. Wash 4X with DFH Add 50 ml/well anti-mouse IgG:HRP diluted in DFH Incubate for 1 hr at 37o C. Wash 3X with DFH and 2X with HBSS. Add 50 ml/well working substrate solution 0.5 ml 4.0% OPD 50 ml 30% H2 O2 1.0 ml 10X Substrate buffer 8.5 ml di-H2 O. Incubate for 20 minutes at room temperature. Add 25 ml/well 4.5N Sulfuric Acid Read A490Notes Test all supernatants at 1:5 dilution. Test ascites at 1:100 |
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