Hc pl fluotar l 20x 0.40 dry objective

All samples were imaged in a Leica DMI6000 B (Leica Microsystems, Wetzlar, Germany) coupled to a workstation running Leica Application Suite X (LASX, Leica Microsystems) with a motorized stage with a HC PL FLUOTAR L 20x/0.40 DRY objective (Cat. No.: 11506243, Leica Microsystems) at an imaging rate of one frame every 8 s for 1 h at 37 °C, without CO₂. The generated movies were exported as *.mov files. These files were analyzed with the Automated Cellular Analysis System (ACAS, Metavi-Harmony software, MetaVi Labs, Austin, TX, USA; sales@metavilabs.com). The evaluation interval was set to 30 s, the minimum track duration to 60 s, the movement threshold to 8 μm and the microscopy resolution to 0.458716 pixel/μm.

The GPx1 knockout cell line (KO.HAP-1.GPx1, cat # HZGHC003261c1010) and its parental HAP-1 strain (cat # C631) were obtained under license from Horizon Discovery (Cambridge, UK). Cells were grown in IMDM medium supplemented with 10% FCS, 1% penicillin (10,000 U/mL) + streptomycin (10 mg/mL) and 1% stable glutamine in a humidified incubator with 5% CO₂ atmosphere. Cells were transferred weekly into new culture flasks when confluence reached 75% (7500 cells in 5 mL medium into a T₂₅ flask) to ensure growth in exponential phase. Once a week, the medium in culture was replaced by fresh one. Cell cultures were routinely tested for mycoplasma. Microscopy was performed with a DMi8 inverse microscope (Leica Microsystems, Wetzlar, Germany) fitted with an HC PL Fluotar L 20x/0.40 dry objective in a transmitted light phase contrast.