Streptomycin

Mouse splenic CD4⁺ T lymphocytes were isolated using a mouse CD4⁺ T Cell Isolation Kit (Miltenyi Biotec; Auburn, CA, USA) with magnetic beads following the manufacturer’s instructions. Mandibular BM-MSCs at passage 2–4 from control mice or periodontitis mice after the indicated manipulation of gene expression were seeded on 24-well plates at a concentration of 1 × 10⁵ cells/well and incubated overnight before they were co-cultured with activated mouse CD4⁺ T cells. Mouse T lymphocytes were pre-activated in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 50 μM 2-mercaptoethanol, 10 mM HEPES, 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin (all reagents were from Thermo Fisher Scientific) by plate-bound anti-CD3 antibody (1:1000 dilution; #ab16669; Abcam, USA) and soluble anti-CD28 antibody (1:5000 dilution; #ab283860, Abcam, USA) for 2–3 days in 24-well plates. Recombinant human TGF-β1 (2 ng/mL; R&D Systems, Minneapolis, MN, USA) and IL-2 (2 ng/mL; R&D Systems) were added into the culture medium to induce Treg differentiation; while Recombinant human TGF-β1 (2 ng/mL; R&D Systems) and IL-6 (50 ng/mL; R&D Systems) were added to induce Th17 differentiation. The activated mouse CD4⁺ T cells were loaded on the adherent BM-MSCs and co-cultured for further 2–3 days. The supernatant and T cells on top layers were collected for further analyses.

CD8+ T cells from the spleens of WT mice were isolated using MACS beads (Miltenyi Biotec) and stimulated for 24 h in 12-well plates (∼3 × 10⁶ cells/well) coated with anti-CD3 (4 µg/mL, Bio X Cell) and anti-CD28 (4 µg/mL, Bio X Cell) in RPMI-1640 media (Hyclone) supplemented with 10% FCS (Hyclone), penicillin (100 U/mL) and streptomycin (100 μg/mL) (Thermo Fisher Scientific), 12 mM Hepes (Thermo Fisher Scientific), and 50 µM β-mercaptoethanol (Sigma-Aldrich). Tumor cells were seeded into 96-well plates (104 cells/well) in DMEM supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL), and IFN-γ (20 ng/mL, abcam) for 24 h for activation. Then, 10⁴ stimulated T cells were seeded onto activated tumor cells for 44 h before counting [28 (link),29 (link)].

BC MCF7 cells were fostered in high-glucose DMEM (Abcam, Shanghai, China) added with 100 U/mL penicillin, 100 mg/L streptomycin, and 10% FBS (Abcam, Shanghai, China) under 5% CO₂, floating in serum-free DMEM/F12 culture medium (Abcam) with 20 ng/mL EGF, 20 ng/mL b-FGF, and 2% B27 (Abcam). Then, we spent 10 days in culturing them within ultra-low attachment plates. miR-30d-5p mimics were synthesized within the GenePharma company (Shanghai, China). Additionally, they were transfected into cells using Lipofectamine 2000 reagent in Invitrogen (USA) in light of the methods of Liu, et al. [16 (link)].

Human normal breast epithelial cell line (MCF-10A) and two human BC cell lines (MCF-7, SKBR-3) were bought from the Chinese Academy of Sciences (Shanghai, China). All cell lines were given culture in RPMI1640 medium (Gibco, U.S.) with 10% fetal bovine serum (FBS, Invitrogen), 100 IU/mL penicillin and 100 mg/mL streptomycin (Abcam, UK) at 37°C in a humid surrounding containing 5% CO₂.