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



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