Saturday, October 18, 2003
Description Bioassay for IFN-gamma Procedure 1) Prepare L929 or A549 cell suspension at a density of 3.5 x 105/ml in assay medium. Add 100 l/well to the 96-well plate and incubate overnight at 37 degrees C, 5% CO2 in a humidified incubator. 2) Dilute samples and standard 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. 3) Aspirate supernatant from the wells carefully and add 100 l/well of EMC virus solution at a density of 1-4 x 104 pfu/ml . 4) Incubate plate for 40 hours at 37 degrees C, 5% CO2 in a humidified incubator. 5) Add 10 l of 5 mg/ml MTT solution to each well and incubate for 4 hours. 6) Add 50 l of MTT Lysing Solution to each well and incubate overnight. 7) Read plate at 570-650 nm 8) Graph standard curve and analyze data. Recipes 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 degrees C -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 Supplies Tips (责任编辑:泉水) |