Ifn γ

To further evaluate the immune response in vivo, BALB/C mice were divided into four experimental groups and immunized two times at an interval of 1 week by intradermal injections of with PBS, MOF, MOF@CM, MOF@DM, and MOF@FM at an equal amount of MOF (50 μL, 3.2 mg mL⁻¹ per mouse). Seven days later after the last vaccination, the peripheral blood which was isolated from mice was centrifuged to obtain the serum. Then the different cytokines in serum were quantitatively analyzed. Briefly, the IL-6 (IL-6, 4 A Biotech Co., Ltd) and IFN-γ (4 A Biotech Co., Ltd) release were detected by ELISA according to the protocol. The spleens were harvested and triturated to obtain a single cell suspension. The cells were filtered through 75 µm filters after washed twice and then the red blood cells were removed by using red blood cell lysis buffer (ACK lysis buffer). Then the T lymphocytes were incubated with anti-CD3-FITC, anti-CD8a-APC, and anti-CD4-PE antibodies after the Fc block of the cells and analyzed by flow cytometry. The lymph nodes were harvested and fixed with 4% paraformaldehyde, embedded with paraffin, and sliced up. After that, the CD3 and CD8 were stained. The images were obtained by an inverted fluorescence microscope.

The human ovarian cancer cell lines SKOV3, OVCAR3, A2780 and Caov3 were obtained from the America Type Culture Collection (ATCC; Manassas, VA, USA), and HO-8910 cell lines, Hosepic cell lines and THP-1 cell lines were obtained from the China Center for Type Culture Collection (CCTCC). The cell lines were cultured in either Dulbecco's modified Eagle's medium (DMEM, HyClone, Cat. No. SH30022.01B) or RPMI-1640 medium (HyClone, Cat. No. SH30809.01B) supplemented with 10% foetal bovine serum (FBS, HyClone, Cat. No. SH30256.01B) and antibiotics (penicillin 100 U/mL, streptomycin 0.1 mg/mL and amphotericin B 0.25 lg/mL) and maintained in a 37 °C incubator containing 5% CO₂. THP-1 cells were treated with 10 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma Aldrich, Germany) for 72 h to induce differentiation of M0 macrophages. M0 macrophages were treated with 100 ng/ml LPS (#L2880, Sigma) plus 10 ng/ml IFN-γ (#BEK-2026, 4A Biotech, Beijing) for 48 h to induce M1 phenotype differentiation or with 10 ng/ml IL-4 (#214–14, PeproTech, Germany) for 48 h to induce M2 phenotype differentiation. Moreover, M0 macrophages were also treated with the supernatant of each ovarian cancer cell line for 48 h. Cells were collected for flow cytometry analysis, RT-qPCR or Western blot assays.