Tnf α duoset

SGHEC-7 cells (1 × 10⁶) were incubated with pooled (n = 9) normal-RI or high-RI dNK conditioned medium for 24 h. The median gestational age was 11.6 weeks (range 9.4–13.9) for normal-RI and 10.7 weeks (range 9.4–13.7) for high-RI cells used to generate pools of conditioned medium, p = 0.3, t-test. Cells were washed three times with PBS and lysed in RIPA buffer. The assay was carried out according to the manufacturer's instructions, using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (TNFα Duoset: R&D Systems, UK and TNFα ELISA development kit: Peprotech, UK).

RAW264.7 cells (1 × 10⁵ cells/well) were seeded in 12-well plates and cultured in D-MEM containing 10% FBS for 24 h followed by serum starvation for 24 h. Then, RAW264.7 cells were stimulated with LPS (100 ng/mL) with or without sealer extracts for 24 h. Proinflammatory cytokines in the culture medium were measured by enzyme-linked immunosorbent assay kits (interleukin [IL]-6 and tumor necrosis factor [TNF]-α DuoSet, R&D Systems, Minneapolis, MN, USA) following the manufacturer's protocol. 3,3′,5,5′-tetramethylbenzidine (SureBlue TMB Microwell Peroxidase Substrate, KPL, Milford, MA, USA) and sulfuric acid (0.6 N) were used as a chromogenic substrate and a reaction stop solution, respectively. Color intensity was measured with a microplate reader (Sunrise) at OD450.

Quantities of interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) in supernatants of BMDC cultures were examined using commercial enzyme-linked immunosorbent assay (ELISA) kits: mouse IL-6 DuoSet (R&D Systems, Minneapolis, MN) and TNF-α DuoSet (R&D System) according to the manufacturer's instructions. The supernatants were diluted 1 : 2 and ran as duplicates. The ODs were measured at 490 nm as described above. Standard curves were plotted and used to calculate the cytokine concentration (pg/ml) in the supernatants.

PBMCs (5 × 10⁵) were incubated in a round-bottom 96-well plate (Corning) at 37°C in a 5% CO₂ incubator in complete RPMI, supplemented with either 200 ng/mL LPS (Sigma-Aldrich), 1 μg/mL Pam3CSK4 (Sigma-Aldrich), α-mannan (see below), β-glucan (see below), or heat-killed (incubation at 70°C for 1 hour) C. cassiicola, which was added at a 1:12 dilution to the cells. After 48 hours, PBMCs were pelleted and the supernatant was collected and stored at –80°C until analysis. Cytokine levels were analyzed via conventional ELISA (Figure 5B; TNF-α DuoSet, R&D Systems, DY210) or Luminex (Figure 1F). Luminex analysis was done via a multiplex bead array assay with antibodies and cytokine standards (R&D Systems, Peprotech). Individual Luminex bead sets (Luminex) were coupled to cytokine-specific capture antibodies according to the manufacturer’s protocols and biotinylated polyclonal antibodies were used at twice the recommended concentrations for a classical ELISA according to the manufacturer’s instructions. The assay was run with 1,200 beads per set of cytokines in a volume of 50 μL. The plates were read on a Luminex MAGPIX platform where more than 50 beads were collected per bead set. The median fluorescence intensity of the beads was then measured for each individual bead, which was analyzed with the Millipex software using a 5P regression algorithm.