Cell viability was detected by Cell Counting Kit-8 (CCK-8) kits. HepG2 cells were seeded onto cultured in 96-well plates at 1 × 104 cells per well. After treatment with or without HG, CCK-8 reagents were added to the plates and then incubated at 37°C for 2 h. A multifunctional microplate reader measured the absorbance at 450 nm (Varioskan LUX, Thermo Fisher Scientific).
HepG2 cells were seeded onto 6 cm cell culture dishes at 2 × 105 cells. Calcein-AM (40719ES50, YEASEN Biotechnology) was applied to measure the intracellular labile iron pool (LIP) [28 (link)]. After treatment, cells were digested and incubated with 0.25 μM Calcein-AM at 37°C for 15 min. Washing with PBS, the fluorescence intensity (Ex/Em = 490 nm/515 nm) was quantified by a multifunctional microplate reader microplate. The intracellular lipid ROS levels were detected by BODIPY 581/591 C11 probe (D3861, Thermo Fisher Scientific) [29 (link)]. Cells were incubated with a 2 μM probe for 37°C for 15 min in the dark. After washed with PBS, the fluorescence spectrum was measured by a multifunctional microplate reader microplate.
HepG2 cells were seeded onto 6 cm cell culture dishes at 2 × 105 cells. Calcein-AM (40719ES50, YEASEN Biotechnology) was applied to measure the intracellular labile iron pool (LIP) [28 (link)]. After treatment, cells were digested and incubated with 0.25 μM Calcein-AM at 37°C for 15 min. Washing with PBS, the fluorescence intensity (Ex/Em = 490 nm/515 nm) was quantified by a multifunctional microplate reader microplate. The intracellular lipid ROS levels were detected by BODIPY 581/591 C11 probe (D3861, Thermo Fisher Scientific) [29 (link)]. Cells were incubated with a 2 μM probe for 37°C for 15 min in the dark. After washed with PBS, the fluorescence spectrum was measured by a multifunctional microplate reader microplate.
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