Antibody Neutralization of TNFa-Induced Killing of L929 Cell

Monday, May 03, 2004

Description
Antibody Neutralization of TNFa-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 g/ml stock aliquot kept at minus 80C (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 3.5x105/ml in assay medium. Add 100 l/well to the 96-well Assay Plate and incubate overnight at 37C, 5% CO2 in a humidified incubator.
Add 50 l/well of Assay Medium to each well of another dilution plate.
Dilute Functional Grade (FG) anti-cytokine antibody by 2-fold serial dilution in the plate from row 3 to 12. Leave rows 1 and 2 blank (control rows).
Add 50 l/well of samples and recombinant cytokine (see Quick Guide Chart for concentration) to the dilution plate from row 2 to row 12 (row 2 will be indicator cells and cytokine). Leave row 1 blank (cells alone).
Incubate the dilution plate at 37C, 5% CO2 in a humidified incubator for 2 hours.
Transfer 50 l/well of the Ab/Ag mixed solution to the corresponding well of the Assay Plate containing cells.
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 l of this working solution of Actinomycin D to each well.
Incubate plate for 24 hrs at 37C, 5% CO2 in a humidified incubator.
Add 10 l/well of 5 mg/ml MTT solution to each well and incubate for 4 hours.
Add 50 l 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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Antibody Neutralization of TNFa-Induced Killing of L929 Cell