Bodipy 581 591 c11 fluorescent probe

Lipid ROS levels in S. aureus, E. coli, and S. pullorum stimulated by ferrous iron were detected using a BODIPY 581/591 C11 fluorescent probe (Invitrogen). Single colonies of S. aureus, E. coli, and S. pullorum on LB solid agar plates were randomly selected and inoculated in 5 mL of LB liquid medium and cultured at 37 °C for 12 h under 180 rpm rotation. The next day, 50 µL of the bacterial inoculation was diluted with 4 950 µL of fresh LB medium and cultured at 37 °C for 3 h at 180 rpm until OD600 reached 0.8. The bacterial precipitate (1 mL) was obtained by centrifugation at 3 600 rpm for 5 min at 4 °C and diluted with H₂O (900 μL). Then, 100 μL of Fe²⁺ (25, 50, 100, and 200 µM) was mixed with S. aureus (900 μL), E. coli (900 μL), and S. pullorum (900 μL) at 37 °C for 30 min, respectively. The bacterial precipitate was obtained by centrifugation at 3 600 rpm for 5 min at 4 °C, after which 200 µL of BODIPY 581/591 C11 probe (2 µM) working solution was added to the bacteria for 30 min of incubation at 37 °C. The lipid ROS level was measured by a multi-scan spectrum with excitation at 488 nm and emission at 525 nm.

The expression level of Fcnb and the number of neutrophils and macrophages were detected by flow cytometry [40 ]. Lung tissue samples were digested to obtain cell suspension. Blood, BALF, and cell suspension were centrifugally stratified to obtain neutrophils and macrophages for re-suspension. Antibodies of LY-6G (11-9668-82, eBioscience, USA) and F4/80 (11-4801-82, eBioscience, USA) were added respectively to the suspensions of neutrophils and macrophages and incubated for 30 min away from light. Then, the mixture of Fcn B antibody (Ab70814, Abcam, UK) and Alexa Fluor 594-goat anti-rabbit IgG (A-11012, Invitrogen, USA) was added to the above cell suspensions and incubated for another 30 min away from light. After washing and suspension, the cells were detected by flow cytometry (A00-1-1102, Beckman, USA). In addition, ROS levels in lung tissues and MLE-12 were detected by flow cytometry. First, the tissue and cells were digested to obtain cell suspensions. The cell suspension was incubated with BODIPY™ 581/591 C11 fluorescent probe (A-11012, Invitrogen, USA) at 37 °C for 30 min and finally detected by flow cytometry.

To assess lipid peroxidation, we first induced ferroptosis in TCMK-1 cells using sodium iodate (SI) with co-treatment of three different OFBP doses. Lipid peroxidation levels were then measured using the BODIPY™ 581/591 C11 fluorescent probe (Invitrogen, CA, USA). Briefly, cells were incubated with 5 μM BODIPY™ probe in fresh culture medium for 30 min at 37 °C. Subsequently, the cells were fixed with 4% paraformaldehyde (PFA) to preserve their morphology. Fluorescence intensity, indicative of lipid oxidation levels in TCMK-1 cells, was measured using a Calibur flow cytometer. The data were analyzed with BD CellQuest™ Pro Software.