Antibody Neutralization of Cytokine-induced Proliferation of

Monday, May 03, 2004

Description
Antibody Neutralization of Cytokine-induced Proliferation of Indicator Cell Lines

Procedure
Materials
Indicator cell line (see Quick Guide Chart for a given cytokine)
Culture Medium (RPMI supplemented with 10% FBS)
Assay Medium (RPMI supplemented with 10% FBS)
96-well flat-bottom culture plate (Costar Cat. No. 3595)
MTT solution (Sigma Cat. No. M5655) 5mg/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
24-48 hour incubation (see Quick Guide Chart)
1 hour assay preparation
Method
Add 50 祃/well of Assay Medium to each well of the 96-well Assay plate.
Dilute Functional Grade (FG) anti-cytokine antibody by 2-fold serial dilution in the Assay plate from row 3 to 12. Leave rows 1 and 2 blank (control rows).
Add 50 祃/well of samples and recombinant cytokine (see Quick Guide Chart for concentration) to the assay plate from row 2 to row 12 (row 2 will be indicator cells and cytokine). Leave row 1 blank (cells alone).
Incubate the plate at 37癈, 5% CO2 in a humidified incubator for 2 hours.
Wash indicator cells 3 times with RPMI1640 and resuspend in Assay Medium at a density of 2-3.5x105 cells/ml (see Quick Guide Chart).
Add 100 祃 of cell suspension to each well.
Incubate cells for 24-48 hours (see Quick Guide Chart) at 37癈, 5% CO2 in a humidified incubator.
Add 10 祃/well of 5 mg/ml MTT solution to the plate and incubate for 4 hours.
Add 50 祃/well of MTT Lysing Solution to the plate and incubate overnight.
Read plate at 570-650 nm.
Graph standard curve and analyze data.

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Antibody Neutralization of Cytokine-induced Proliferation of