Bodipy 581 591 c11

After treatments, 1.0 × 10⁶ cells per experimental group were stained with BODIPY 493/503 (Sigma, 1 μM) or BODIPY 581/591 C11 (Sigma, 2 μM) [22 (link)]. BODIPY 581/591 C11 was added to the 6-OHDA treatment. BODIPY 493/503 was added to cultures 15 min before finishing the incubation with 6-OHDA. Then, cells were washed with PBS 1X, collected by trypsinization, and diluted in 0.5 mL PBS 1X in flow cytometry tubes. Finally, samples were analyzed in a FACS Canto II flow cytometer, and light emissions at different wavelengths of stained cells were measured (530/30 and 582/15 filters for BODIPY 581/591 C11 and 530/30 filter for BODIPY 493/503). Data from cell aggregates, dead cells, and cell debris signals were removed.

The level of lipid peroxidation was assessed using a fluorescent probe of BODIPY581/591‐C11 (Sigma‐Aldrich, St Louis, MO, USA).^[23] A suspension of 1 × 10⁶ cells mL⁻¹ and mFeS‐or Fe²⁺‐treated H1N1 virus was loaded with the probe at a final concentration of 2 µm. The suspension was then incubated at 37 °C for 30 min, washed by centrifugation to remove the unbound probe, and detected by flow cytometry (Beckman Coulter, Brea, CA, USA) for intracellular level of lipid peroxidation. The level of lipid peroxidation (green) induced by mFeS‐or Fe²⁺‐treated H1N1 viruses was observed using a fluorescent microscope (Leica, Tokyo, Japan).

Staining with BODIPY 493/503 (Sigma, 1 μM) and BODIPY 581/591 C11 (Sigma, 2 μM) was performed prior to fixation or flow cytometry analysis. BODIPY 581/591 C11 was added to the 6-OHDA treatment. BODIPY 493/503 was added to cultures 15 min before finishing the incubation with 6-OHDA. Then, immunocytochemistry or flow cytometry analysis was performed.