Toll-Like Receptor 4 (TLR4) has been identified next to MD2 and CD14 as a receptor that is central to the innate immune response to lipopolysaccharides (LPS) of Gram negative bacteria. TLR4 contributes to the initiation of CNS neuroimmune activation after nerve transection. TLR4 is part of an early, specific, innate CNS/microglial response and maybe relevant in the prevention and treatment of neuropathic pain syndromes. TLR4 is weakly expressed by resting cells, but is upregulated following stimulation with LPS.
Figure 1: Lipopolysacharide (LPS) induces IL-8 secretion in HEK293 cells transfected with hTLR4/hMD-2 (293-hTLR4/hMD-2) in a dose dependant manner.
Figure 2: To measure the ability of this antibody to neutralize the TLR4-mediated LPS response on 293-hTLR4/hMD-2 cells, various concentrations of this antibody were incubated with 293-hTLR4/hMD-2 cells in a 96-well plate for 1 hour at 37° C. Following this preincubation period, LPS was added. The assay mixture, in a total volume of 100 μL, containing anti-hTLR4 at the concentrations indicated, LPS at 75 ng/mL, and cells at 5 x 105 cells/mL, were incubated at 37° C for 20 - 24 hours in a humidified incubator. After this incubation, supernatant was collected from each well and tested for hIL-8 levels using the Human IL-8 Quantikine ELISA kit (for example, R&D Systems, Catalog # D8000C). The ND50 of this antibody under these conditions is approximately 1.5 - 7.5 μg/mL.
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