Monday, May 03, 2004
Description TNF-alpha-Induced Killing of L929 Cell Line Procedure Materials L929 mouse fibroblast line (ATCC Cat. No. CCL-1) Culture Medium (RPMI supplemented with 10% FBS) Assay Medium (RPMI supplemented with 2% FBS) 96-well flat-bottom culture plate (Costar Cat. No. 3595) Actinomycin D, 500 礸/ml stock aliquot kept at minus 80癈 (protect from light) 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 48-hour incubation (see Quick Guide Chart) 1 hour assay preparation Method Prepare L929 cell suspension at a density of 3.5 x 105/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. Dilute samples and standard (see Quick Guide Chart for standard range) by 2-fold serial dilution in the Assay Medium in 100 祃/well in another 96-well plate from row 2 to 12. Leave row 1 as blank. Prepare a 4 礸/ml working solution of the Actinomycin D by diluting the 500 礸/ml stock 125 times in the Assay Medium. Keep Actinomycin D solution protected from light. Add 50 祃 of this working solution of Actinomycin D to each well. Transfer 50 祃 of titrated samples and standard to the corresponding wells on the Assay Plate. Incubate plate for 24 hrs at 37癈, 5% CO2 in a humidified incubator. Add 10 祃/well 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) |