Liperfluo

Live cell staining with Liperfluo (Dojindo, Kumamoto, Japan; #L248) was performed according to the manufacturer’s protocols. Briefly, these dyes were diluted in PBS to 1 μM, added to the cell pellets, and incubated in the dark at 37 °C for 10–20 min. The supernatant was removed by centrifugation and the cells were washed three times with PBS. The stained cells were then resuspended in an appropriate volume of PBS and seeded on 4-well glass bottom dishes (Φ9.5 mm/well, MATSUNAMI, Bellingham, WA USA; #D141400). Using a confocal laser scanning microscope (LMS880, Carl Zeiss, Oberkochen, Germany), Liperfluo-stained cells were visualized by acquiring lipid peroxide-derived fluorescence spectra (520–550 nm) in the lambda mode and merging them with the transmitted light image to visualize their localization. Further, the positivity and cell diameter of Liperfluo-stained cells were quantified by BD FACSVerse™ Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA). For HE staining, cell suspensions were applied to coated glass slides, allowed to stand for 30 min, immediately fixed in 99% ethanol for at least 5 min, washed with water, and stained with H&E. Images were visualized under a BX63 microscope (40× magnification; Olympus, Tokyo, Japan) and calculated using ImageJ software.

Lipid peroxidation was determined by measuring the malondialdehyde (MDA) level with a lipid peroxidation MDA assay kit (Beyotime Biotechnology). Briefly, cells were homogenized and the supernatant was mixed with thiobarbituric acid (TBA)-glacial acetic acid reagent. Then, the TBA-MDA mixture was heated at 100 °C for 1 h. The absorbance was measured at 532 nm with a microplate reader and the MDA concentration was calculated as μM/mg protein. Lipid peroxidation was also confirmed by fluorescence observations using Liperfluo (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. After an incubation with the indicated treatments, cells were stained with 5 μmol/L Liperfluo and Hoechst nuclear stain and then visualized under a florescence microscope (Carl Zeiss, Oberkochen, Germany).