Apc anti mouse mhc 2

For the in vivo targeting study, male C57BL/6 mice (20 ± 2 g) were subcutaneously injected with Fluc mRNA-loaded 1.5PD-LNPs, PC-LNPs, and SAPC-LNPs (10 μg/mouse, n = 3). D-fluorescein potassium (3 mg/100 μL) was injected intraperitoneally at 24 h. The popliteal lymph nodes of the mice were collected for bioluminescence imaging 10 min later using a Xenogen IVIS Spectrum Imaging System (Perkin Elmer, USA).
Another group of male C57BL/6 mice weighing 20 ± 2 g were subcutaneously injected with EGFP mRNA loaded into 1.5PD-LNPs, PC-LNPs, SAPC-LNPs, or PBS at a dose of 10 μg/mouse. The popliteal lymph nodes of the mice were collected to prepare single cell suspensions. The lymph nodes were gently ground on 70 μm cell mesh strainers and then centrifuged at 4 °C for 10 min at a speed of 500 g. The cells were precipitated and resuspended in PBS. For each test, APC anti-mouse MHC-II (eBioscience) and PE anti-mouse CD11c (eBioscience) antibodies were added according to the protocol and incubated for 30 min at 4 °C. The cell precipitate was resuspended in PBS for flow cytometry analysis. The sections were stained with primary antibodies against CD169, followed by immunostaining using PE-conjugated secondary antibodies and DAPI staining to visualize the nuclei.

The surface markers expressed in the stimulated DCs were assessed by using a FACS method. The proportion of DCs expressing co-stimulatory surface markers was determined in CD11c-gated DCs. DCs (1 × 10⁶) pulsed with STG, LPS, or PBS as a negative control were stained with the following monoclonal antibodies: FITC anti-mouse CD11c (eBioscience, USA; clone N418e), APC anti-mouse MHC-II (eBioscience; clone M5/114.15.2), and CD80 and PE anti-mouse CD40 (eBioscience; clone 1C10). The stained cells were washed three times with FACS running buffer (Miltenyi Biotec, Germany). Subsequently, the antigen-pulsed DCs were sorted with a MACSQuant Analyzer (Miltenyi Biotec).