Navios flow cytometer

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).

Data acquisition and data analysis were conducted at the Cochin Cytometry and Immunobiology Facility. For iron assay measurements using BioTracker Far-red Labile Fe2+ Dye (called FerroFarRed, #SCT037, Sigma; Thermofischer; Waltham, MA), 2 × 10⁵ cells were washed twice in PBS to remove extracellular Fe2+ then labeled with 2 µM Sirhonox for 60 min in a tissue culture incubator (37 °C, 5% CO²) in the dark and then washed twice again. The pelleted cells were then resuspended in 0.3 mL of PBS. FCM data were collected using a BD Navios flow cytometer. 10,000 events were recorded for analysis. Data analysis was then carried out with KALUZA software. For lipid peroxide production measurements using C11- BODIPY (581/591) (2 mM) (Thermofischer, Waltham, MA), 2 × 10⁵ cells were labeled with C11-BODIPY in 1 mL of warm complete medium for 10 min in a tissue culture incubator (37 °C, 5% CO²) in the dark. Cells were then washed twice and resuspended in 200 µL of fresh PBS. FCM data were collected using a C6 Accuri flow cytometer (Becton Dickinson, Le Pont de Claix, France) with CFlow Plus software. 10,000 events were captured for subsequent analysis with CFlow Plus software (Becton Dickinson, Le Pont de Claix, France).