*2.8. Comparison of Transgene Expression Activity*

At the tissue level, C57BL/6N mice were intravenously injected with Nluc mRNAloaded A-11-LNPs, MC3-LNPs or RNA-LPX at a dose of 0.5 mg mRNA/kg. Twenty-four hours after the injection, liver, spleen, and inguinal lymph node (LN) were harvested, frozen in liquid nitrogen, and stored at −80 ◦C for use in a Nluc assay, as described in Section 2.7.

For splenic DC level, C57BL/6N mice were intravenously injected with EGFP mRNAloaded A-11-LNPs, MC3-LNPs or RNA-LPX at a dose of 0.5 mg mRNA/kg. Twenty-four hours after the injection, the spleen was harvested. Splenocytes were dissociated from the remaining spleen tissues and were passed through a cell strainer (40 µm pore). The recovered cells were spun down (400× *g*, 4 ◦C, 5 min), and the supernatant was discarded. 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 1 <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 and then incubated at 4 ◦C for 10 min. The resulting material was then treated with an APC anti-mouse CD11c antibody and a PerCP/Cyanine 5.5 anti-mouse I-A/I-E antibody at 4 ◦C for 30 min. The resulting cells were washed with FACS buffer twice, filtered via a nylon filter, and stained with PI, and the DCs were then analyzed for EGFP and I-A/I-E expression using CytoFLEX (Beckman Coulter, Inc., Brea, CA, USA).
