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


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