Liperfluo

Mouse sperm (2 × 10⁶ sperm) were collected from the cauda epididymis and washed with PBS thrice, and then incubated with a fluorescence probe in darkness. Liperfluo (Dojindo), indicator of lipid peroxidation, was added to each sample at a final concentration of 2 μm and incubated for 30 min at 37 °C. A Si‐DMA kit (Dojindo) evaluated mitochondrial singlet oxygen (¹O₂) levels in samples that were incubated at a final concentration of 50 nm for 45 min at 37 °C. For sperm analysis of the cellular ROS and mitochondrial membrane potential (MMP), DCFH‐DA (Yeasen, Shanghai, China; magnification = 1000×), JC‐1 (Yeasen; magnification = 200×), and TMRM (M20036, Invitrogen, Waltham, MA, USA; magnification = 1000×) probes were incubated at 37 °C for 30 min. After incubation, samples were washed twice and resuspended in PBS for flow cytometry assay (Cytoflex S flow cytometer, Beckman, CA, USA). A total of 10 000 events were collected for measuring fluorescence intensity.
Meanwhile, after fluorescent staining, 20 μL of a sperm suspension was smeared on a clean slide glass and covered with 22 mm square‐shaped coverslips. Fluorescent staining of Liperfluo (Ex: 488 nm) and si‐DMA (Ex: 640 nm) in sperm were also recorded under an upright fluorescent microscope (Zeiss 710, Jena, Germany).

Harvested at the density of 10 × 10⁶, H9C2 cells were stained with H2DCFDA (MedChemExpress, China) to detect ROS level, and stained with Bodipy (BODIPY™ 581/591 C11, Invitrogen, USA) or Liperfluo (Dojindo, Shanghai, China) to detect lipid peroxidation level. The signals were collected using by flow cytometry (CytoFLEX S equipped with Kaluza analysis 2.1, Beckman coulter, USA) or imaged by an Olympus FV3000 confocal laser scanning microscope (Olympus, Japan).

The cells were washed and resuspended in phosphate-buffered saline containing 3% FBS, followed by incubation with fluorescent-conjugated antibodies specific for CD49d (clone: 9F10), CD71 (clone: GA-R2), and CD235a (clone: M-A712) (Becton Dickinson). Propidium iodide (PI; Thermo Fisher Scientific) was used for the marking of dead cells. Liperfluo (Dojindo, Kumamoto, Japan) was used for the detection of cellular lipid peroxide. FerroFarRed (Goryo Chemical, Sapporo, Japan) was used for the detection of intracellular labile iron. Data were acquired with FACS Aria II or FACS Canto II (Becton Dickinson) and were analyzed using FlowJo version 7.6.5 software (TreeStar, Ashland, OR, www. flowjo. com).

Lipid peroxidation was detected via Liperfluo (Dojindo, Kumamoto, Japan). Cells were incubated with 5 μM Liperfluo for 30 min at 37 °C in accordance with the manufacturer’s instructions. After removing Liperfluo, EESCs were washed three times with HBSS. Fluorescence microscopy (Nikon, Tokyo, Japan) was used to obtain fluorescence images from 3 separate dishes for each treatment.

Lipid ROS, considered one of the most important markers of ferroptosis, was measured by using Liperfluo (#L248, Dojindo, Kumamoto, Japan). We seeded cells in 24-well plates, pretreated them with erastin-containing medium for 24 hours, and incubated them for 30 minutes in the dark with Liperfluo at a working concentration of 1 μM. PBS was then used to wash the cells three times. Finally, a fluorescence microscope (Olympus, Tokyo, Japan) was used to acquire images at a magnification of ×40.