Anti-TLR4 Neutralization of LPS-induced Cytokine Production

Monday, May 10, 2004

Description
Toll-like receptor 4 (TLR4) and its associated MD-2 play a role in the response of myeloid cells to LPS. While the exact mechanism is not clearly defined at the present time, antibodies to TLR4 have been reported to inhibit the LPS-induced cytokine production by human PBMCs and mouse peritoneal macrophages. We have slightly modified the blocking experiments published in the references below with monoclonal antibodies HTA125 (anti-human TLR4) and MTS510 (anti-mouse TLR4/MD2). This protocol provides a general guideline; investigators should optimize different experimental conditions including cell type and number, concentrations of antibody, proper type of stimulation, and incubation times in their own assay systems.

Procedure
Prepare human PBMCs, dispense 1ml at 2x106/ml into each well of a 24-well culture plate.
Pre-incubate cells with 20 礸/ml of HTA125 or isotype control antibody.
Incubate for 0.5-1 hour at 37癈.
Add LPS at a concentration of 10 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 human IL-6 or TNF-a Ready-SET-Go ELISA set:

Recipes
Materials
Human PBMC suspension
Culture Medium (RPMI supplemented with 10% FBS)
24-well flat-bottom culture plate (Costar Cat. No. 3526)
Functional Grade (FG) anti-human TLR4, clone HTA125 (Cat. No. 16-9917)
Functional Grade (FG) mouse IgG2a isotype control (Cat. No. 16-4724)
Lipopolysaccharide (LPS) (Sigma, Cat. No. L-8274)
eBioscience Human IL-6 Ready-SET-Go ELISA set (Cat. No. 88-7066), or
eBioscience Human TNF-a Ready-SET-Go ELISA set (Cat. No. 88-7346)
Experiment Duration
1 hour assay preparation
16-24 hour incubation
Time required for ELISA Ready-SET-Go

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Anti-TLR4 Neutralization of LPS-induced Cytokine Production