C11 bodipy581 591

Cells were seeded in 6-well dishes before the experiment. After OGD/R treatment, the cells were harvested by trypsinization and washed with Hanks Balanced Salt Solution (HBSS; #88284, Invitrogen). The cells were resuspended in 500 μL of HBSS containing 2 μM C11-BODIPY (581/591) (#D3861, Invitrogen) and incubated for 10 min at 37 °C in an incubator with 5% CO₂. The cells were then washed and resuspended in 500 μL of HBSS, strained through a 40 μm cell strainer and analyzed by flow cytometry (CytoFLEX, Beckman Coulter) or microplate reader.

Levels of intracellular ROS and NO and extent of lipid peroxidation in primary murine keratinocytes were determined using H₂DCF-DA (Invitrogen/Life Technologies), DAF-FM-DA (Sigma), or C11-BODIPY^581/591 assays (Invitrogen/Life Technologies), respectively. H₂DCF-DA allows detection of intracellular H₂O₂, but it also detects oxygen radicals [52 (link)]. DAF-FM-DA is a probe to detect NO, but it only works under aerobic conditions and it is likely to react with an oxidative product of NO, rather than with NO itself [53 (link)]. Cells were incubated for 30 min with 50 μM H₂DCF-DA or 5 μM DAF-FM-DA, or for 2 h with 2 μM C11-BODIPY^581/591 in cell culture medium at 37°C prior to detachment by trypsin. Fluorescence was directly measured by flow cytometry using the BD Accuri C6 (BD Biosciences, San Jose, CA).

To measure cell death, cells were stained by SYTOX^® Green (S11368, Invitrogen, Carlsbad, CA, USA) to a final concentration of 1 μM for 20 min and then analysed by flow cytometry. Lipid ROS generation was measured by adding C11‐BODIPY 581/591 (D3861, Invitrogen) to a final concentration of 1.5 μM for 30 min before flow cytometry. MitoROS production was evaluated by incubating cells with MitoSOX^® red mitochondrial superoxide indicator (M36008, Thermo Fisher) to a final concentration of 5 μM for 10 min before flow cytometry. JC‐1 (C2006, Beyotime) was used to indicate changes of MMP, as measured by flow cytometry.

First, HepG2 and NCTC cells were seeded in 6‐well plates at a density of 1.0 × 10⁵ cell well⁻¹, and were then cultured overnight. Afterward, the cells were pretreated with 100 µm FeCl₃ for 3 h, followed by washing with PBS for three times, and further treated with different compounds at 20 µm for 12 h. After washing with PBS for three times, cells were stained with Ca‐AM at 10 µm at 37 °C for 30 min. Finally, after washing with PBS for three times, cells were re‐suspended in PBS for flow cytometry analysis on an Novocyte Flow Cytometer (USA).
To assess lipid peroxidation, HepG2 and NCTC cells were exposed to 100 µm FeCl₃ and incubated for 12 h at 30 °C. Pretreated cells were washed with PBS for three times and then treated with DFO, DFX, and DFA1 at the concentration of 20 µm for 12 h. After washing with PBS for three times, cells were stained with 10 µm C11‐BODIPY^581/591 (Invitrogen, USA) for 2 h. The treated cells were washed three times by PBS and re‐suspended for flow cytometry analysis on an Novocyte Flow Cytometer (USA).

Cells were plated at a density of 3 × 10⁵ cells in 2 ml medium per well in six-well plates (Corning Falcon). The next day, the medium in each well was replaced with 2 ml fresh medium with compounds for treatment. 18 h later, the cells in each well were washed with PBS once, then incubated with 1 ml PBS containing 2 μM C11-BODIPY 581/591 (Invitrogen) at 37 °C for 15 min. After a brief PBS rinse, the cells were trypsinized and resuspended in 500 μl PBS and analyzed in an Attune NxT flow cytometer (Thermo Fisher Scientific) with the BL1 detector using the 488 nm laser line. A minimum of 10,000 single cells were analyzed per sample.

The novel marine bromophenol compound, XK-81 (Purity ≥ 98%), was supported by the Institute of Oceanology of the Chinese Academy of Sciences (Qingdao, China). Roswell Park Memorial Institute 1640 (RPMI1640), Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum (FBS) were purchased from Life Technologies/Gibco Laboratories (Grand Island, NY, USA); erythrocyte lysis buffer was purchased from Gefan Biotechnology (Shanghai Gefan Biotechnology, Shanghai, China); the Mito-FerroGreen kit was purchased from Dojindo (Kumamoto, Japan); C11 BODIPY 581/591 were purchased from Invitrogen (Camarillo, CA, USA); an immunohistochemistry detection system kit was bought from Bioss (Beijing, China); antibodies for GAPDH, CD80, CD206, GPX4, SLC7A11, and ACSL4 were purchased from ABclonal Technology (Wuhan, China). ELISA kits for IL-12, IL-1β, TNF-α, and IFN-γ were obtained from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China); all other chemicals were purchased from Sigma Aldrich (St. Louis, MI, USA).