Monday, May 10, 2004

Description
Anti-TLR4/MD2 Neutralization of Lipid A-induced Cytokine Production by Mouse Peritoneal Cells

Procedure
Prepare peritoneal cells and dispense 1ml at 2x106/ml into each well of a 24-well culture plate.
Pre-incubate cells with 20 礸/ml of MTS510 or isotype control antibody.
Incubate for 0.5-1 hour at 37癈.
Add Lipid A at a concentration of 5 ng/ml.
Incubate cells for 16-24 hours at 37癈, 5% CO2 in a humidified incubator.
Harvest 1ml of media from each well, spin at 500x g, collect supernatant and discard the cell pellet.
Follow the protocol for mouse IL-6 or TNF-a Ready-SET-Go ELISA set:

Recipes
Materials
Thioglycolate elicited mouse peritoneal cell suspension
Culture Medium (RPMI supplemented with 10% FBS)
24-well flat-bottom culture plate (Costar Cat. No. 3526)
Functional Grade (FG) anti-mouse TLR4/MD2, clone MTS510 (Cat. No. 16-9924)
Functional Grade (FG) rat IgG2a isotype control (Cat. No. 16-4321)
Lipid A (Sigma, Cat. No. L-0774)
eBioscience Mouse IL-6 Ready-SET-Go ELISA set (Cat. No. 88-7064), or
eBioscience Mouse TNF-a Ready-SET-Go ELISA set (Cat. No. 88-7324)
Experiment Duration
1 hour assay preparation
16-24 hour incubation
Time required for ELISA Ready-SET-Go

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