5 6 carboxyfluorescein diacetate succinimidyl ester cfse

For analysis of antigen presentation ability, WT and KO mice were immunized by footpad inoculation with keyhole limpet hemocyanin (KLH; FujiFilm Wako, Osaka, Japan) mixed with Freund’s complete adjuvant (Nacalai Tesque, Kyoto, Japan). At 7–10 days after inoculation, the immunized mice were euthanatized under anesthesia with Isoflurane (FujiFilm Wako). Whole lymphocytes were obtained by disruption of popliteal and inguinal lymph nodes. The cell suspensions were depleted of erythrocytes by hypotonic lysis at room temperature. CD4⁺ T-cells were isolated using a CD4⁺ T-cell isolation kit II (Miltenyi Biotec) in accordance with the supplied manual, using auto-MACS (Miltenyi Biotec). CD4⁺ T-cells were stained with 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Sigma-Aldrich) for the detection of cell proliferation. BMDCs from WT and KO mice were stimulated with KLH for 16 h. After incubation, the KLH-stimulated DCs (5 × 10³) were co-cultured with KLH-primed CD4⁺ T-cells (5 × 10⁵) in a 96-well U-bottom plate. After 4 days of incubation, CD4⁺ T-cells were collected and stained with anti-CD4 antibody (PE) (Biolegend) and 7-amino-actinomycin D (7AAD) (Becton Dickinson). The stained cells were analyzed using a MACSQuant flow cytometer (Miltenyi Biotec).

Flow cytometry-based cytotoxicity assays were performed using K562 cells as targets and primary NK cells treated with 10 µM PRO or 5 nM BRY or not treated (nt) for 18 h following a previously described method (43 (link)) with minor modifications. Briefly, NK cells were labeled with 5 µM 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE; Sigma-Aldrich) for 7 min at 37°C, washed twice, then seeded with 2 × 10⁵ K562 cells at different effector-to-target cell (E:T) ratios in a 96-well plate for 4 h at 37°C 5% CO₂. Then, cells were labeled with 5 µg/ml 7-aminoactinomycin D (7-AAD; Sigma-Aldrich) for 20 min at +4°C, washed, and fixed with 1% PFA. For each E:T ratio, 20,000 target cells (gated as CFSE⁻) were acquired by FACS. The percentage of specific lysis was calculated as follows: 100 × (% 7-AAD⁺ target cells in sample − basal % 7-AAD⁺ target cells)/(100 − basal % 7-AAD⁺ target cells). The specific lysis derived from three E:T ratios per sample was conversed to lytic units (LU), defined as the number of effector cells required to lyse 20% of 2 × 10⁵ target cells (44 (link)), and results were expressed as the number of LU contained in 10⁷ NK cells.

Bone marrow-derived dendritic cells (BM-DCs) were prepared for the phagocytosis assay. Bone marrow cells were isolated from the femurs of mice and red blood cells were lysed following treatment with ammonium chloride potassium lysis buffer at room temperature for 3 min. Cells at a concentration of 1 × 10⁶ cells/ml were cultured in complete medium composed of RPMI-1640 medium supplemented with recombinant mouse IL-4 (10 ng/ml) and recombinant mouse GM-CSF (50 ng/ml) (PeproTech, USA) for 7 days, and then the non-adherent cells (BM-DCs) were harvested for co-culture with irradiated tumor cells.
MC38-OVA and EG7-OVA cells were subjected to 5 or 10 Gy of radiation (or sham-irradiation). The irradiated cells were incubated for 48 h in complete medium and then stained with 3 μM 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Sigma-Aldrich, USA). BM-DCs were harvested and stained with anti-mouse CD11c at 4°C for 30 min, followed by co-culture with CFSE-labeled MC38-OVA or EG7-OVA in a 96-well-plate for 4 h. The phagocytosis of irradiated tumor cells was analyzed by flow cytometry. For microscopy analysis of cell engulfment, DCs and tumor cells that were cultured and stained as described above were mounted on a coverslip and assessed with a fluorescence microscope (Life Technologies, USA) at a magnification of 200×.