*2.9. Measurement of Activation Markers in Splenocytes*

C57BL/6N mice were intravenously injected with OVA mRNA-loaded A-11-LNPs, MC3-LNPs or RNA-LPX at a dose of 0.03 mg mRNA/kg. Spleen tissues were harvested, and the resulting dissociated splenocytes were passed through a cell strainer (40 µm pore) 24 h after the injection. The recovered cells were spun down (400× *g*, 4 ◦C, 5 min), the supernatant discarded, and the resulting cells were resuspended in RBC Lysis buffer (1 mL) and incubated for 5 min at room temperature. These treated cells were washed with HBSS(-) by spinning (400× *g*, 4 ◦C, 5 min). The concentration of cells was adjusted to <sup>1</sup> <sup>×</sup> <sup>10</sup><sup>7</sup> cells/mL with FACS buffer, and the resulting cells were treated with a 10 µg/mL solution of an anti-mouse CD16/32 antibody, followed by incubation at 4 ◦C for 10 min. This preparation was then treated with fluorophore-conjugated antibodies at 4 ◦C for 30 min. Cells were washed with FACS buffer twice, filtered via nylon filter, stained with PI, and applied for flowcytometric analysis (CytoFLEX). DCs, T cells, B cells, and NK cells were defined as PI−I-A/I-E+CD11c<sup>+</sup> cells, PI−CD3+CD19<sup>−</sup> cells, PI−CD3−CD19<sup>+</sup> cells, and PI−CD3−CD19−NK1.1<sup>+</sup> cells, respectively. Relative expression of CD40, CD80, CD86 in DCs and CD69 in B cells, T cells, and NK cells were measured.
