C11 bodipy 581 591

The level of cellular lipid peroxidation was detected by a C11-BODIPY 581/591 (Cayman Chemical, Michigan). After the cells were treated with the test compounds, 5 μM of C11-BODIPY 581/591 dye was added to fresh medium and incubated in the dark at 37 °C for 30 min. The C11-BODIPY 581/591 dye solution was removed, and then the cells were washed twice with PBS to remove the excess probe. Lipid ROS fluorescence was captured by a Leica laser scanning confocal microscope. Three random fields per sample were quantified using ImageJ software.

Intracellular ROS and lipid peroxide levels were measured using DCFH-DA (Beyotime, Cat#S0033 M) and C11-BODIPY 581/591 (Cayman Chemical, Cat#217075-36-0), respectively. Mitochondrial ROS and mitochondrial peroxidized lipid levels were measured using MitoSOX Red (Life Technologies, Cat#M36008) and MitoPerOx (Cayman Chemical, Cat#18798), respectively. Briefly, 2.5 × 10⁵ cells were seeded into each well of 12-well plates and cultured overnight. The next day, cells were treated with ML162 for 2–3 h, followed by adding probes into the culture medium for 30 min at 37 °C. After washing with PBS, cells were harvested by trypsinization, resuspended in 400 μL PBS, and analyzed using a flow cytometer (BD LSRFortessa). Data analysis was performed using FlowJo software. For fluorescence microscopy-based analysis, cells were seeded into a 35 mm confocal dish and cultured overnight. Following drug administration, cells were stained with probes for 30 min at 37 °C, and then images were documented using a laser scanning confocal microscope (Leica TCS SP8 DLS) to analyze intracellular and mitochondrial ROS and lipid peroxide levels in live cells.

Apoptotic cell staining was performed with an Annexin V-FITC and propidium iodide kit (e-Bioscence, San Diego, CA) in n = 8 primary CLL samples following the manufacturers’ protocol.
Healthy PBMCs were stained with a viability dye (780 Invitrogen, e-Bioscience) and anti-CD5 PC7 and anti-CD19 ECD antibodies (Beckman Coulter, Brea, CA, USA). To detect the presence of lipid oxidation indicating ferroptosis, CLL cells were stained in Hank’s balanced salt solution (HBSS) for 10 min at 37 °C with 5 μM C11-BODIPY 581/591 (#27086, Cayman Chemical, Ann Arbor, MI, USA), and the mean fluorescence intensity was detected by flow cytometry.
To investigate the presence of reactive oxygen species (ROS), CLL cells were stained with viability dye and with the cell-permeant 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (D399, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) by diluting the reagents to a final working concentration of 1 µM in prewarmed PBS and incubating for 15 min at RT.
All experimental data were acquired with BD Navios flow cytometer (BD Pharmingen), and the results were analyzed using FCS Express 7 software (De Novo software).

H9c2 cardiomyoblasts were seeded in 24 mm coverslips coated with 0.1% collagen and incubated with NT containing 2 μM C11 BODIPY581/591 (Cayman Chemical Company) and Hoechst 33342 for 30 min at 37°C. Images were captured at 40x magnifications using a confocal microscope and analyzed using NIS-Elements imaging software. For cardiomyocytes, the cells were seeded in 24 mm 0.1% collagen-coated coverslips. After 24 h, the cells were incubated with NT containing 2 μM C11 BODIPY581/591 for 1 h at room temperature. Images were captured at 40x magnifications using a confocal microscope and analyzed using NIS-Elements imaging software.

After the heat shock treatment (50 °C), cells were stained with 5 µM H₂DCFDA (Sigma, D6883) for the detection of ROS accumulation, 5 µM C11-BODIPY™ 581/591 (Cayman, 27086) for the detection of lipid peroxidation, 3 µM JC-1 (Cayman, 10009172) for staining the mitochondrial membrane potential, 15 µm Calcium Green-1, AM (Cayman, 20400) for staining cytosolic calcium, and 1 µM FerroOrange (Merck, SCT210) for iron staining at room temperature for 20–30 min, followed by two washes with 1X PBS. Fluorescence images were captured using a Leica fluorescence microscope (Leica Microsystems, Germany) equipped with a Leica DFC9000 sCMOS camera. The excitation/emission of fluorescent dyes used the following filters: H₂DCFDA (Ex/Em: 492/515 nm), C11-BODIPY™ 581/591 (Ex/Em: 460–495/510–550 nm), JC-1 (Ex/Em: 488/538 nm for monomers and 596 nm for oligomers), Calcium Green-1 (Ex/Em: 506/531 nm), and FerroOrange (Ex/Em: 543/580 nm). For fluorescence intensity, 100 µL of stained cells were transferred into a black 96-well plate and fluorescence was measured using a Synergy HTX Multi-mode Plate Reader (BioTek, VT, USA) with the indicated excitation and emission wavelengths.