Accuri c6 1

BALB/c mice (6–8 weeks) were subcutaneously transplanted with 1 × 10⁶ 4T1 tumour cells on the flank. The mice were treated with different formulations and irradiated for 30 min with or without a 665 nm laser at an intensity of 0.15 W/cm². The treatments were performed three times every other day. Lymph nodes were surgically isolated 72 h after different treatments. The samples were then mechanically disrupted and digested in a solution containing 0.5 mg/mL collagenase IV at 37 °C for 40 min. Suspensions were filtered and washed with PBS containing 2% FBS. Single-cell suspensions were then stained with anti-CD11c-PE, anti-CD80-FITC and CD86-APC antibodies for 30 min. Flow cytometry analysis was performed. Spleens were surgically isolated 7 days after the final treatment. The samples were mechanically disrupted and incubated with ammonium chloride buffer to lyse erythrocytes and incubated with anti-CD3-PE and anti-CD8-APC before flow cytometry (BD Accuri C6 1.0.264.21, BD, USA).

For flow cytometry analysis, immature BMDCs were treated with PBS, rHAD (10 μg/mL) and HAD (10 μg/mL) for 12 h. After that, BMDCs were collected and stained with antibodies and analysed by flow cytometry. Meanwhile, the IL-6 and TNF-α concentrations in the culture supernatant were determined using ELISA kits. Alternatively, immature BMDCs were co-incubated with the residues of 4T1 tumour cells using a transwell system. The 4T1 cells were incubated with PBS, free Ce6, DP-Ce6, or LIA (Ce6 concentration 2.5 μg/mL) with or without irradiation (665 nm, 0.15 W/cm², 2 min). Then BMDCs were incubated with the residues of 4T1 cells for 12 h. The cells were stained with antibodies and analysed by flow cytometry (BD Accuri C6 1.0.264.21, BD, USA).