Sandwich type elisa

Thioglycollate-elicited peritoneal cells were collected from wild-type (WT), ΔTLR2, and ΔTLR4 male C57BL/6 mice at 6 weeks of age provided kindly from Prof. Suzanne Michalek (University of Alabama at Birminghum). Mouse primary peritoneal cells or RAW264.7 cells were dispensed into 96-well plates (SPL, Suwon, Korea) at a density of 2 × 10⁵ cells/ml and stimulated with TIGR4 or TIGR4Δlgt for 12 h. For nitrite measurement, 100 μl of culture supernatant was mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, and 2% phosphoric acid) and incubated at RT for 5 min. The optical density was then measured at a wavelength of 540 nm using a Victor X3 light plate reader (Perkin-Elmer, Waltham, MA, USA). NaNO₂ solution was used to generate the standard curve. The amount of TNF-α in the cell culture supernatant was determined with a commercially available sandwich-type ELISA (eBioscience) according to the manufacturer's protocol.