Aurora 3 laser flow cytometer

For PDT in mono-culture, 4 × 10⁴ MC38, or 1 × 10⁵ D1DCs were seeded in 24-well plates in their respective culture media and incubated overnight at 37 °C and 5% CO₂. Cells were then incubated with ZnPc-EVs with a ZnPc concentration calibrated at 4 µM for 4 h. Cells were then washed 3 times with PBS and supplied with 500 µL fresh medium. Illumination was performed at a light intensity (fluence rate) of 200 mW/cm² for a total light dose (fluence) of 20 J/cm² using a 690 nm LED Laser (CNI lasers, Changchun, China). Cells were then incubated for 18 h, collected in FACS buffer, stained with 0.5 µM DAPI in FACS buffer and analyzed by flow cytometry on a Cytek Aurora 3-Laser flow cytometer. As a positive control, cells were subjected to three freeze/thaw cycles at −20 °C (FT) before staining and analysis by flow cytometry on a Cytek Aurora 3-Laser flow cytometer (Cytek, Fremont, USA). For PDT in co-culture, 3 × 10⁴ MC38CFP cells were seeded in 24-well plates and incubated overnight at 37 °C and 5% CO₂. The next morning, the cells were counted and an equal amount of D1DCs was added. Cells were then incubated with the ZnPc-EVs with a ZnPc concentration calibrated at 4 µM for 4 h, treated with PDT or FT, and analyzed as described.