Cytometric bead array assay

In a random subset of 15 patients, whole blood was stimulated ex vivo with LPS on day 1 of ICU admission, as previously described [20 (link)]. Heparin-anticoagulated blood was stimulated for 3 h at 37 °C in pyrogen-free RPMI 1640 (Life Technologies, Bleiswijk, the Netherlands) with or without 100 ng/mL ultrapure LPS (from Escherichia coli 0111:B4; InvivoGen, Toulouse, France). TNF-α and IL-1β were measured in supernatants using a cytometric bead array assay (BD Biosciences, San Jose, CA, USA). Cytokine release was calculated as the difference in cytokine levels in samples incubated with and without LPS. The medical ethical committee of the Academic Medical Center in Amsterdam gave ethical approval for the study (number NL34294.018.10). Written informed consent was obtained from all patients, or their legal representative, and from healthy volunteers.

RAW264.7 cells were plated in a 96-well plate at a density of 1 × 10⁵ cells per well in 100 μL of culture media and incubated for 12 h. Subsequently, the differentiated RAW264.7 cells were treated with PM_2.5 at different concentrations of 12.5, 25, 50, 100, and 200 μg/mL for an additional 12 and 24 h. The supernatant of the PM_2.5 extract-treated cells was collected to assess TNF-α secretion using an enzyme-linked immunosorbent assay (ELISA) obtained from eBioscience (San Diego, CA, USA).
Further, other cells were pretreated with BTE at 0, 2.5, 5, 10, and Coelonin at 0, 1.25, 2.5, 5, and 10 μg/mL for 2 h before treating with 200 μg/mL PM_2.5 water extract, in another group, same treatments by BTE and Coelonin were repeated and followed by treating with 100 μg/mL organic extract. After 18 h treatment, the supernatant of these cells was then collected for the analysis of TNF-α, IL-6, and MCP-1 secretion using an ELISA and a BD Biosciences cytometric bead array assay (San Jose, CA, USA). Each immunoassay was performed in accordance with the manufacturer’s instructions.