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Monday, May 03, 2004
Description IFN-gamma Protection from Viral Infection of 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 summary chart) 1 hour assay preparation Method Prepare L929 or A549 cell suspension at a density of 3.5 x 105/ml in assay medium. Add 100 祃/well to the 96-well plate and incubate overnight at 37癈, 5% CO2 in a humidified incubator. Dilute samples and standard (see Quick Guide Chart for standard range) by 2-fold serial dilution in the Assay Plate from row 2 to 12. Leave row 1 as blank. 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 1-4 x 104 pfu/ml (see Quick Guide Chart for human IFN-gamma: 1/250 dilution of stock of 107 pfu/ml, for mouse IFN-gamma, 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) |
IFN-gamma Protection from Viral Infection of Cell Lines
时间:2006-07-24 14:10来源:Scienceboard.net 作者:admin
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