Monday, May 03, 2004

Description
IFN-gamma Protection from Viral Infection of Cell Lines

Procedure
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癈
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
Method
Prepare L929 or A549 cell suspension at a density of 3.5 x 105/ml in assay medium. Add 100 祃/well to the 96-well 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 Plate from row 2 to 12. Leave row 1 as blank. Incubate the Assay Plate for 6 hours.
Aspirate supernatant from the wells carefully and add 100 祃/well of EMC virus solution at a density of 1-4 x 104 pfu/ml (see Quick Guide Chart for human IFN-gamma: 1/250 dilution of stock of 107 pfu/ml, for mouse IFN-gamma, 1/1000 dilution of stock of 107 pfu/ml).
Incubate plate for 40 hours at 37癈, 5% CO2 in a humidified incubator.
Add 10 祃 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.

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