Spectral cell analyzer

Female C57BL/6J mice (5~6 weeks old, CLEA Inc.) were subcutaneously inoculated with Lewis lung carcinoma cells (8×10⁴ cells/mouse) into their left flanks on day 0. On days 4, 7, and 10 post tumor inoculation, the following substances in 100μL saline were subcutaneously injected into the right flanks of mice according to the divided groups: 1) saline; 2) dLLC (6 μL autologous tumor antigens/mouse in free format); 3) dLLC-ML (Large-size metal organic framework encapsulated with autologous tumor antigens: 6 μL/mouse autologous tumor antigens and 600 μg/mouse particles); 4) dLLC-MS (Small-size metal organic framework encapsulated with autologous tumor antigens: 6 μL/mouse autologous tumor antigens and 600 μg/mouse particles). Tumor size was measured by a caliper and tumor volume was calculated according to the formula: 1/2 × length × width².
At the endpoint, spleen was harvested and triturated through a 70 μm cell strainer to obtain single cell suspension. The cells were stained with anti-CD3-APC, anti-CD4-PE/Cyanine7, anti-CD8a-APC/Cyanine7, anti-CD44-FITC and anti-CD62L-PE antibodies (Biolegend) after the Fc block using purified anti-CD16/CD32 antibody. Flow cytometry was performed using a Spectral Cell Analyzer (SP6800, Sony) and data analysis was carried out with FlowJo software.

Recombinant EBV (EBV-GFP) was generated from EBV-positive Burkitt lymphoma cells (EBV+Akata) and a cell-free infection was conducted^{19 (link),20 (link)}. EBV with GFP integrated was produced from Akata cells based on a previous study^{19 (link)} with slight modifications. Briefly, Akata-EBV cells were crosslinked with 0.8% (v/v) goat anti-human IgG (Bersee) for 6 h; then the cells were cultured for 72 h with standard medium. EBV particles were collected by centrifugation at 54,000 × g for 2 h and resuspended with serum-free DMEM. The virus suspension was aliquoted in 0.5 ml aliquots and stored at −80 °C or used immediately for infection studies. For cell-free infection, NP69, HK1 and HEK293 were exposed to EBV for 3 h before detecting the number of copies of the viral genome. For detection of the infection efficiency, cells were cultured for an additional 72 h, after which the medium was replaced. The infection efficiency was roughly judged under a fluorescence microscope (Leica) and precisely measured using a flow cytometer (Spectral Cell Analyzer, Sony) on the basis of GFP expression.

Female C57BL/6J mice (CLEA Inc.) were immunized by subcutaneously injecting Fe- based cancer vaccines into the left flank (Fe-based metal organic framework particles, 600 μg/mouse; F-OVA, 100 μg/mouse). An equivalent dose of F-OVA in free format was used as control. Immunized mice were euthanized one day later, and nearby draining lymph nodes were harvested. To analyze the lymph node targeting, the obtained lymph nodes were freshly frozen in Tissue-Tek O.C.T. compound to prepare the cryostat sections. Then, the sections were mounted using SlowFade™ Diamond mountant with DAPI and observed using Leica CLSM. To carry out the analysis about antigen cross-presentation, the obtained lymph nodes were ground through a 70 μm cell strainer to obtain single cell suspension. The obtained cells were blocked with purified anti-CD16/CD32 antibody, and then stained with anti-CD11c-APC and anti-H-2K^b- SIINFEKL-PE (BioLegend) antibodies. Flow cytometry was performed using a Spectral Cell Analyzer (SP6800, Sony). FlowJo software was used for the analysis of flow cytometry data.