*2.8. Cytokine and Chemokine Secretion Analysis*

To evaluate cytokine and chemokine production, BMMΦs were seeded at 0.2 <sup>×</sup> <sup>10</sup><sup>6</sup> cells/well in sterile 24-well plates and incubated with 300 µg/mL of the different iNP formulations at 37 ◦C and 5% CO<sup>2</sup> for three hours. Excess iNPs were removed by washing twice with PBS followed by replacing with complete medium containing 100 ng/mL LPS or 200 ng/mL CpG ODN. After 48 h, cell culture supernatants were collected and analyzed using enzyme-linked immunosorbent assays (ELISA) (BioLegend) to measure murine interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) or Luminex (Austin, TX, USA) Multi-Analyte Profiling technology (xMAP) to assess multiple cytokines and chemokines as described in the text.
