Quantification of Cytokines and TNF-α in Cell Supernatants

Cytokines were quantified in AM supernatants using a Milliplex MAP Human Cytokine Kit (Millipore, #HCYTOMAG-60K) as detailed in Hoppstädter et al. (23 (link)). AMs were kept at a density of 1 × 10⁵ cells per well in 96 well plates in a total volume of 100 μL medium in the presence or absence of Pam₃CSK₄ (100 ng/mL) or Poly(I:C) (10 μg/mL) for 6 h. The supernatants were collected and stored at −80°C until they were used in the multiplex cytokine assay. The immunoassay procedure was performed using a serial dilution of the 10,000 pg/mL human cytokine standard according to the manufacturer's instructions, and the plate was read at the Luminex 200 System (Millipore).
Murine TNF-α was quantified by bioassay as previously described (11 (link)). L929 cells were seeded at a density of 3 × 10⁴ cells per well into a 96-well plate. After 24 h, the medium was replaced by 100 μL of actinomycin D solution (1 μg/mL in standard medium) and cells were incubated for 1 h at 37°C. Subsequently, BMM supernatants (100 μL per well) were added. Dilution series of recombinant murine TNF-α (100–2,500 pg/mL) were run alongside the samples to generate a standard curve. The plates were incubated for an additional 24 h at 37°C. The MTT assay (see Determination of Cell Viability) was used to assess cell viability.