Saturday, October 18, 2003
Description TNF-alpha Bioassay Procedure 1) Prepare L929 cell suspension at a density of 3.5 x 105/ml in assay medium. Add 100 l/well to the 96-well Assay 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 Medium in 100 l/well in another 96-well plate from row 2 to 12. Leave row 1 as blank. 3) Prepare a 4 g/ml working solution of the Actinomycin D by diluting the 500 g/ml stock 125 times in the Assay Medium. Keep Actinomycin D solution protected from light. Add 50 ul of this working solution of Actinomycin D to each well. 4) Transfer 50 l of titrated samples and standard to the corresponding wells on the Assay Plate. 5) Incubate plate for 24 hrs at 37 degrees C, 5% CO2 in a humidified incubator. 6) Add 10 l/well of 5 mg/ml MTT solution to each well and incubate for 4 hours. 7) Add 50 l of MTT Lysing Solution to each well and incubate overnight. 8) Read plate at 570-650 nm. 9) Graph standard curve and analyze data. Recipes 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 g/ml stock aliquot kept at minus 80 degrees C (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 1 hour assay preparation Supplies Tips (责任编辑:泉水) |