B16 blue ifn α β reporter cells

Infections were performed in 12-well plates. Supernatants from infected cells were collected at the indicated time points in the figure legends, and spun down at 12,000 × g for 5 min to remove any debris. TNF-α (#900-K54), IL-1β (#900-K47), IL-10 (#900-K53) and IP-10 (CXCL10) (#250-16) in the supernatants were quantified using ABTS ELISA Development Kit (PeproTech) according to the manufacturer’s instructions. All experiments were performed in duplicate, and three independent experiments were conducted.
For quantification of type I IFN (INF-α/β) in the supernatants of iBMDMs, cells were infected for 16 h, and supernatants were collected. Murine type I IFNs were detected using B16-Blue IFN-α/β reporter cells (InvivoGen) which carry an SEAP reporter gene under the control of the IFN-α/β-inducible ISG54 promoter and that have an inactivation of the IFN-γ receptor. Supernatants from iBMDM cells were incubated with the reporter cell line, and levels of SEAP in the supernatants were determined using the detection medium QUANTI-Blue (InvivoGen) after 24 h as per the manufacturer’s instructions using recombinant mouse IFN-β (PBL Assay Science, catalogue number 12401-1) as a standard. Experiments were run in duplicates and repeated at least three times. Results are expressed as OD at 655 nm.

Human PBMCs or mouse BM cells were plated on 96-well plates at 1 × 10⁶ cells/well/200 μL. D35 (1 μM = 6.3 μg/mL) and DOTAP-Nano/-Lipo or D35+DOTAP-Nano/-Lipo were added to the cell cultures overnight at 37°C in a CO₂ incubator. The centrifuged supernatant was collected and used for cytokine ELISA. Human IFN-α was measured with human IFN-α pan-ELISA development kit (Mabtech). Mouse IFN-α/β (type I IFN) production was measured using B16-Blue IFN-α/β reporter cells (InvivoGen).