Human peripheral blood mononuclear cells (PBMC, Zen-bio, Research Triangle Park, NC) were washed twice in RPMI 1640 and resuspended in complete culture media at a concentration of 5 × 106 cells/mL. 100 μL of cell suspension was seeded into 96-well plate (5 × 105 cells/well) and treated with PBS, 200 nM siRNA, RNP8, PEI/siRNA complex, or LPS (10 μg/mL) and PHA (20 μg/mL). The cell culture supernatants were collected after 24 h and 72 h incubation. Cytokines were analyzed using LEGENDplex™ Human Cytokine immunoassay (Biolegend, San Diego, CA).
To evaluate potential in vivo immune responses, immune competent C57BL6 mice were injected with PBS, siRNA (2mg/kg), RNP8 or LPS (2 μg per mouse) intravenously. Two hours post administration, mice were sacrificed and the blood was collected for cytokine analysis using LEGENDplex™ mouse Cytokine immunoassay kit (Biolegend, San Diego, CA).
To evaluate potential in vivo immune responses, immune competent C57BL6 mice were injected with PBS, siRNA (2mg/kg), RNP8 or LPS (2 μg per mouse) intravenously. Two hours post administration, mice were sacrificed and the blood was collected for cytokine analysis using LEGENDplex™ mouse Cytokine immunoassay kit (Biolegend, San Diego, CA).