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Monday, May 03, 2004
Description Antibody Neutralization of IFNg-Protection from Viral Infection of L929 and A549 Cell Lines Procedure Materials L929 mouse fibroblast line (ATCC Cat. No. CCL-1) or A549 human lung carcinoma (ATCC Cat. No. CCL-185) Culture Medium (RPMI supplemented with 10% FBS) Assay Medium (RPMI supplemented with 2% FBS) 96-well flat-bottom culture plate (Costar Cat. No. 3595) EMC virus, 107 pfu/ml aliquots stock kept at minus 80癈 MTT solution (Sigma Cat. No. M5655) 5 mg/ml stock in PBS kept at room temperature (protect from light) MTT Lysing solution, 20% SDS/50% DMF Instruments Pipettes and pipettors Humidified incubator 96-well micro test spectrophotometer Experiment Duration 24 hour incubation (see Quick Guide Chart) 1 hour assay preparation Method Prepare L929 or A549 cell suspension at a density of 3.5x105/ml in assay medium. Add 100 祃/well to the 96-well Assay Plate and incubate overnight at 37癈, 5% CO2 in a humidified incubator. Add 50 祃/well of Assay Medium to each well of anther dilution plate. Dilute Functional Grade (FG) anti-cytokine antibody by 2-fold serial dilution in the plate from row 3 to 12. Leave rows 1 and 2 blank (control rows). Add 50 祃/well of samples and recombinant cytokine (see Quick Guide Chart for concentration) to the dilution plate from row 2 to row 12 (row 2 will be indicator cells and cytokine). Leave row 1 blank (cells alone). Incubate the dilution plate at 37癈, 5% CO2 in a humidified incubator for 2 hours. Transfer 50 祃/well of the Ab/Ag mixed solution to the corresponding well of the Assay Plate containing cells. Incubate the Assay Plate for 6 hours. Aspirate supernatant from the wells carefully and add 100 祃/well of EMC virus solution at a density of 1-4x104 pfu/ml (see Quick Guide Chart for human IFN-g: 1/250 dilution of stock of 107 pfu/ml, for mouse IFN-g, 1/1000 dilution of stock of 107 pfu/ml). Incubate plate for 40 hours at 37癈, 5% CO2 in a humidified incubator. Add 10 祃 of 5 mg/ml MTT solution to each well and incubate for 4 hours. Add 50 祃 of MTT Lysing Solution to each well and incubate overnight. Read plate at 570-650 nm Graph standard curve and analyze data. Recipes Supplies Tips (责任编辑：泉水) |
Antibody Neutralization of IFNg-Protection from Viral Infect
时间:2006-07-24 14:10来源:Scienceboard.net 作者:admin
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