A1r live cell confocal microscope

The mRNAs encoding SARS-CoV-2 antigens, RBD and S, were formulated by TT3 nanoparticles to 0.03 and 0.06 mg mL⁻¹, respectively. The formulated nanoparticles were dialyzed in 1 × PBS. The formulation was injected intramuscularly to female C57BL/6 mice with 1.5 μg RBD-encoding mRNA or 3.0 μg S-encoding mRNA. Mice were euthanized 6 h after injection. The muscle tissue at the injection sites was harvested and incubated in 4% paraformaldehyde overnight, followed by another 30% sucrose incubation overnight. The tissue was sectioned using Cryotome E Cryostat (Thermo-Shandon) and fixed using acetone. The subsequent staining procedure was the same as that for staining 293T cells. Images were taken on a Nikon A1R Live Cell Confocal microscope with NIS-Elements BR imaging software (version 4.20).

To monitor peptide internalization, 1 mL of HeLa cell suspension (5 × 10⁴ cells) was seeded in a 35-mm glass-bottomed microwell dish (MatTek) and cultured overnight. Cells were gently washed with DPBS twice and treated with 5 μM FAM-labeled peptide in phenol red-free DMEM containing 1% FBS for 37 °C in the presence of 5% CO₂. After 2 h of incubation, the medium was removed, and the cells were gently washed with DPBS twice and phenol red-free DMEM was added. The cells were imaged on a Nikon A1R live-cell confocal microscope equipped with 100X oil objective in a heated chamber at 37 °C and 5% CO₂. Images were captured under the same parameters. NIS-Elements AR was used to denoise the images and add scale bars. Images presented in this report were generated using the instruments and services at the Campus Microscopy and Imaging Facility, The Ohio State University.