Cd86 fitc clone gl1

The immunostimulatory effects of PDT were investigated by seeding 5 × 10⁴ MC38 cells, 4 × 10⁴ CT26 cells or 3.5 × 10⁴ TC-1 cells in 24-well plates and 10⁴ D1DCs in 96-well plates (Corning, Glendale, CA, USA). After overnight incubation at 37 °C and 5% CO₂, the cancer cells were incubated with 300 pM AU-011 in culture medium for 4 h, and subsequently illuminated with 690 nm light at 400 mW/cm² for 0.25, 2.5, or 25 J/cm². As controls, cells were subjected to incubation with 300 pM AU-011 only, 690 nm light only or three freeze/thaw cycles at  − 20 °C (FT). Following this, the PDT-treated tumor cells were added to the D1DCs at a ratio of 20: 1 (tumor cell: D1DC) and incubated for 24 h at 37 °C and 5% CO₂. Cells were then collected, stained with 0.5 µM DAPI (Sigma-Aldrich, Zwijndrecht, The Netherlands), CD86-FITC (clone GL1; Thermofisher, Landsmeer, The Netherlands) and anti-CD11c-PE (Clone HL3; BD Biosciences, New Jersey, USA) before analysis by flow cytometry on a Cytek Aurora 3-Laser flow cytometer. D1DC controls consisted of D1DCs in mono-culture, poly I:C at 1 µg/mL, 300 pM AU-011 directly incubated with D1DCs, or 300 pM AU-011 incubated with tumor cells added to D1DCs without treatment (dark toxicity).

The immunostimulatory effects of PDT were investigated by seeding 5 × 10⁵ MC38CFP cells in 24-well plates and 10⁴ D1DCs in 96-well plates (Corning, Glendale, CA, USA). After overnight incubation at 37 °C and 5% CO₂, the cancer cells were incubated with the ZnPc-EVs with a ZnPc concentration calibrated at 4 µM for 4 h and treated with PDT or FT as described. The (dying) treated tumor cells were then added to the D1DCs at a ratio of 20: 1 (tumor cell: D1DC) and incubated for 24 h at 37 °C and 5% CO₂. The cells were then collected, stained with 0.5 µM DAPI (Sigma-Aldrich, Zwijndrecht, The Netherlands), anti-CD11c-PE (Clone HL3, BD Biosciences, New Jersey, USA) and CD86-FITC (clone GL1, Thermofisher, Landsmeer, The Netherlands), and finally, analyzed by flow cytometry on a Cytek Aurora 3-Laser flow cytometer. Controls consisted of D1DCs in mono-culture (-), poly I:C at indicated concentration, ZnPc-EVs directly incubated with D1DCs, or ZnPc-EVs incubated with tumor cells added to D1DCs without PDT.