3.10. C11-BODIPY lipid peroxidation assay

Lipid peroxidation was measured by the C11-BODIPY 581/591 (D3861, Invitrogen, USA). The treated cells were incubated with the kit reagent (10 μM in DMEM without FBS) for 30 min. Thereafter, the harvested cells were washed twice with PBS and re-suspended in 500 μl PBS. Lipid peroxidation levels were measured using flow cytometry (BD FACSCalibur) (λ_ex: 488 nm), and the data were analyzed using FlowJo. A total of 10,000 cells in each sample was analyzed.

In addition, the lipid peroxidation was also visualized and measured simultaneously during PDT treatment using confocal laser scan microscope (CLSM) by adapting the fluorescence recovery after photo-bleaching (FRAP) method. Briefly, 150,000 cells were plated at a 35 mm glass-bottom dish and allowed to adhere overnight. Cells were then pretreated with vehicle (DMSO, 4 h) or HYP (2 μM, 4 h) in the dark, before the dish was put on the stage of an inverted confocal microscope equipped with a chamber containing 5% CO₂ atmosphere and heated to 37 °C (Leica TCS SP8). Imaging was carried out using a water-immersion objective HC PL APO 63 × /1.20 W heated to 37 °C. Basel lipid peroxidation level was firstly measured by detecting 500–530 nm emission after 488 nm excitation of oxidized C11-BODIPY. The HYP incubated cells were then excited with the 633 nm laser line using the single iteration of bleaching pulse focused on the selected area of a single cell or a group of cells, to mimic the PDT irradiation. Right after the 633 nm pulse, fluoresce of the oxidized C11-BODIPY was detected with 488 nm excitation and 500–530 nm collection. The area with elevated oxidized C11-BODIPY fluorescence was roughly overlapped with the selected area excited with 633 nm laser. Usually 60 images of oxidized C11-BODIPY were acquired during 60 s or 180 s. Fluorescence intensity from the selected area under 633 nm excitation was then used to quantify the change of oxidized C11-BODIPY level.