Victor x3 light plate reader

Mouse blood samples were taken every 2 weeks. Serum was isolated via centrifugation and stored at −80°C until use. Pneumococcal strains were cultured in THY (THB with 2% yeast extract) and harvested at mid-log phase. The absorbance of the pneumococcal pellet was adjusted to an OD₆₀₀ of 0.1 by dilution with PBS. Then, 96-well immunoplates (SPL) were coated with 100 μl pneumococcal suspension and incubated overnight at 4°C to allow adherence of bacterial cells. The plates were then washed five times with PBS-T, followed by blocking with 1% BSA in PBS for 1 h at RT. After blocking, diluted serum was added to each well and incubated at RT for 1 h, and unbound antibodies were removed by washing with PBS-T. Appropriate dilutions of goat anti-mouse Ig-HRP (Sigma-Aldrich), goat anti-mouse IgG-HRP (Southern-Biotech), or goat anti-mouse IgM-HRP (Southern-Biotech) were added to wells and incubated for 30 min at RT. After washing the plates five times with PBS-T, 100 μl TMB substrate reagent (BD Biosciences, Franklin Lakes, NJ, USA) was added. When colors developed, 50 μl of 2 N H₂SO₄ was added, and the absorbance was measured at 450 nm using a Victor X3 light plate reader (Perkin-Elmer).

Total carbohydrates were estimated using the anthrone method as described previously^{21 (link)}. Lyophilized DeinoPol was dissolved in 0.2 mL of distilled water and mixed with 0.8 mL of 0.2% anthrone solution in sulfuric acid. DeinoPol was hydrolyzed into simple sugars at 90 °C for 10 min and cooled to room temperature. The optical density was then measured at 620 nm using a Victor X3 light plate reader (PerkinElmer, Waltham, MA, USA). d-glucose was used as a standard, and values are represented as percent of dry weight of the samples. The concentration of DNA and protein in purified DeinoPol was measured using the NanoVuePlus spectrophotometer (GE Healthcare; IL, USA).

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.

Cells were plated and treated as for the cytokine analysis, but incubated for 2.5 hours. CM was then harvested by centrifugation and active caspase-1 measured with the Caspase-Glo 1 Inflammasome Assay (Promega) following the manufacturer’s instructions. Luminescence was measured after 90’ with VICTOR X3 Light Plate Reader (PerkinElmer). Luminescence values obtained in the presence of Ac-YVAD-cho inhibitor were subtracted from the non-inhibited reaction, to obtain the caspase-1-specific activation.