Introduction

Toll-like receptor 2 (TLR2) plays a role in the response of myeloid cells to microbial lipoprotein/lipopeptides. Antibodies to TLR2 have been reported to inhibit the lipoprotein/lipopeptide-induced cytokine production by human PBMCs and mouse macrophages. This protocol provides a general guideline; investigators should optimize experimental conditions including cell type, density, antibody concentration, ligands and kinetics.

Anti-TLR2 Neutralization of Cytokine Production by Human PBMC

Materials

• Cell suspension of human target cells of interest
• Culture Medium (RPMI supplemented with 10% FBS)
• 24-well flat-bottom culture plate (Costar Cat. No. 3526)
• Functional Grade (FG) anti-human TLR2, clone TL2.1 (Cat. No. 16-9922)
• Functional Grade (FG) mouse IgG2a isotype control (Cat. No. 16-4724)
• eBioscience Human IL-6 Ready-SET-Go! ELISA set (Cat. No. 88-7066) or eBioscience Human TNF-α Ready-SET-Go! ELISA set (Cat. No. 88-7346)

Experiment Duration

• 1 hour assay preparation
• 14 hour incubation
• Time required for ELISA Ready-SET-Go!

Method

1. Prepare target cells of interest at the concentration of 2x10⁶/ml in 10% FBS-Culture media (or 5% human serum).
2. Pre-incubate the cells with 10-20µg/ml of TL2.1 or isotype control antibody.
3. Incubate for 30 minutes at room temperature.
4. Add the stimuli.
5. Incubate cells for 14 hours at 3°C, 5% CO₂ in a humidified incubator.
6. Harvest 1ml of media from each well, spin at 500xg, collect supernatant and discard the cell pellet.
7. Follow the protocol of eBioscience Ready-SET-Go! ELISA set to detect IL-6 or TNF-α production.

Important: Make sure to prevent LPS contamination in the culture system. Listeria is one of the most potent TLR2 agonists.

Anti-TLR2 Neutralization of Cytokine Production by Mouse Macrophage

Materials

• Cell suspension of mouse target cells of interest (Peritoneal cells or Raw264.7)
• Culture Medium (RPMI supplemented with 10% FBS)
• 24-well flat-bottom culture plate (Costar Cat. No. 3526)
• Functional Grade (FG) anti-mouse/human TLR2, clone T2.5 (Cat. No. 16-9024)
• Functional Grade (FG) mouse IgG1 isotype control (Cat. No. 16-4714)
• eBioscience Mouse IL-6 Ready-SET-Go! ELISA set (Cat. No. 88-7064) or eBioscience Mouse TNF-α Ready-SET-Go! ELISA set (Cat. No. 88-7324)

Experiment Duration

• 1 hour assay preparation
• 24 hour incubation
• Time required for ELISA Ready-SET-Go!

Method

1. Prepare target cells of interest and dispense 1ml at 2x10⁶/ml into each well of a 24-well culture plate.
2. Pre-incubate cells with 50µg/ml of T2.5 or isotype control antibody.
3. Incubate cells for 30 minutes at 37°C, 5% CO₂ in a humidified incubator.
4. Add the stimuli.
5. Incubate for 24 hours at 37°C, 5% CO₂ in a humidified incubator.
6. Harvest 1ml of media from each well, spin at 500xg, collect supernatant and discard the cell pellet.
7. Follow the protocol for mouse IL-6 or TNF-α Ready-SET-Go ELISA set.

References

Meng G, et al. 2004. Antagonistic antibody prevents toll-like receptor 2-driven lethal shock-like syndromes. J Clin Invest 113(10):1473-81.