Co-culture Cytokine Release Assay

For the co-cultures, A549 cells (10⁵ cells/well) were seeded at the bottom and THP-1 cells (10⁵ cells/well) were plated on the insert (0.4 μm pore polyester filter) of Transwell culture plates (#3470, Corning Inc., Corning, NY, USA), with the two cell cultures being physically separated to avoid direct contact, according to the method described by Li et al. [32 (link)]. After 24 h co-culture, the cells were exposed to the following treatments: vehicle (DMSO 0.5% in PBS), CsA_rm 10 µg/mL, CsA_M20 at 10 µg/mL in respect to CsA, mannitol 2 µg/mL in DMSO 0.5% in PBS. After 1 h, LPS 1 μg/mL (Escherichia coli O55:B5; cat# L6529; Sigma Aldrich, Merck, Milano, Italy) was added to the culture and maintained for 24 h. Cells incubated with the vehicle and not exposed to LPS were used as the control. The concentration of IL-6 in the conditioned media was subsequently determined using an ELISA kit (Boster Biological Technology, Milano, Italy; cat. no. IL-6, EK0410), according to the manufacturer’s protocol and expressed as pg/mL.