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 g/ml of MTS510 or isotype control antibody. Incubate for 0.5-1 hour at 37C. Add Lipid A at a concentration of 5 ng/ml. Incubate cells for 16-24 hours at 37C, 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 Supplies Tips (责任编辑:泉水) |