Bodipy 581 591 c11

Lipid peroxidation was measured by flow cytometry with the use of BODIPY 581/591 C11 (Thermo Fisher Scientific). Cells were incubated with drug under normoxic or hypoxic conditions for 18 h. After the addition of BODIPY 581/591 C11 to a final concentration of 5 μM, the cells were incubated for an additional 30 min, isolated by exposure to trypsin, stained for 5 min with PI in DMEM for dead cell exclusion, and then analyzed with an Attune flow cytometer (Thermo Fisher Scientific Inc.) with excitation at 488 nm and a 530/30-nm emission filter to detect oxidized forms of the probe. MFI was calculated with Attune software v2.1.0 (Thermo Fisher Scientific Inc.).

BODIPY 581/591 C11 (Thermo Fisher, Waltham, MA, USA) was dissolved in DMSO at a concentration of 2 mM. After treatment with drugs, the cells were added with BODIPY 581/591 C11 at a final concentration of 2 μM, and then cultured at 37 °C and 5% CO₂ for 20 min. After PBS washing, N2A cells were digested into a single cell with trypsin and analyzed by FACS. The detection channel was FITC (BODIPY 581/591C11 excitation light at 488 nm and emission light at 530 nm).

The ability of SI to induce lipid peroxidation was investigated using lipid peroxidation sensor BODIPY 581/591 C11 (Thermo Fisher Scientific, Waltham, USA). BODIPY 581/591 C11 is a lipophilic fluorescent dye for indexing lipid peroxidation in cellular membranes^{55 (link),56 (link)}. In brief, ARPE-19 cells were seeded in a 96-well plate at density of 8000 cells/well. After 24 h, cells were loaded with 2 μM BODIPY 581/591 C11 for 30 min at 37 °C followed by treatment with or without NaIO₃ for 0.5 h or 6 h. As a positive control, cells were treated with 100 μM tBH and 100 nM Heme for 0.5 h or 6 h. Then the rates of lipid peroxidation were measured using a Varioskan Flash multimode reader (Thermo Fisher Scientific, USA) with excitation/emission of 495/521 nm for the green signal (oxidized) and 575/600 nm for the red signal (non-oxidized). Oxidation of BODIPY 581/591 C11 is presented as a ratio between green fluorescence (oxidized) and total fluorescence (oxidized plus non-oxidized).

The level of lipid peroxidation was determined by measuring the amount of MDA produced by the thiobarbituric acid reaction as described by Campo et al. (2014 ). Briefly, samples (0.05 g) were homogenized in 1 ml of 80% (v/v) ethanol and centrifuged at 16,000g for 20 min at 4 °C. The supernatant (0.5 ml) was recovered and mixed with 0.5 ml of 20% (w/v) trichloroacetic acid containing 0.65% (w/v) thiobarbituric acid. The mixture was incubated at 85 °C for 30 min and then quickly cooled in an ice bath. After centrifugation (10,000g, 10 min), the absorbance of the supernatant was measured at 532 nm and 600 nm (Spectramax M3 reader, Molecular Devices, USA). The value for nonspecific absorption at 600 nm was substracted from the 532 nm reading. The MDA content was calculated using its molar extinction coefficient (156 mM⁻¹ cm⁻¹), and the results are expressed as mM MDA/g fresh weight (FW).
C11-BODIPY^581/591 (Thermo Fisher Scientific) was used to visualize lipid peroxidation (10 µM C11-BODIPY^581/591) following the procedure described by Shen et al. (2020 (link)). Epifluorescence of fluorescent dye was visualized using a Leica DM6 and the fluorescence emitted by oxidized C11-BODIPY^581/591 was observed at 488 nm in an Leica DM6 microscope under GFP fluorescence (488 nm excitation, 509 nm emission).

The lipophilic probe C11BODIPY581/591 (Thermo Fisher Scientific, Waltham, MA, USA) was used to detect lipid peroxidation. Fluorescence changes (i.e., shifts from red to green upon oxidation) indirectly reflect the oxidation of unsaturated fatty acids [32 (link)]. Neuronal cells in the control, M0-Exosome, and M1-Exosome groups were stained with 1 µM C11BODIPY581/591 and Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at 37 °C in a 5% CO₂ incubator and then assessed by laser confocal microscopy (Zeiss LSM800; Zeiss AG; Oberkochen, Germany) according to the manufacturer’s instructions. Lastly, data were quantified using ImageJ software (National Institutes of Health).