The CCK-8 kit only detects indirect cell viability. Almost 5000 WT-ALOX5 or Ser663Ala cells/100 μl were seeded into 96-well-plate per well, cells were treated with erastin for 8 hours in the presence of propofol.
Then, 10 μl CCK-8 reagent was added into per well. After 2 hours incubated with CCK-8 reagent, the indirect viability could be measured by a microplate reader (Molecular Devices, US) at 450 nm. PI/Hoechst 33342 staining ((PI, P4170, sigma, Germany), (Hoechst, #b2261, sigma, Germany)) and CCK-8 (#C0037, Beyotime, China) was used to detect cell necrosis rate in such study. In the PI/HO staining experiment, HT22 cells were seeded in the 24-wells-plates at a density of 1×10 5 / mL. After overnight incubated, pretreated with the indicated concentration of PPF for 2 h before erastin adding. After 8 h treatments of erastin, cells were incubated with 5 μg/mL PI and 10 μg/mL Hoechst 33342 for 15 min. The results were captured by Olympus uorescence microscope. Cell necrosis rate was calculated by PI (+)/Hoechst (+).
Then, 10 μl CCK-8 reagent was added into per well. After 2 hours incubated with CCK-8 reagent, the indirect viability could be measured by a microplate reader (Molecular Devices, US) at 450 nm. PI/Hoechst 33342 staining ((PI, P4170, sigma, Germany), (Hoechst, #b2261, sigma, Germany)) and CCK-8 (#C0037, Beyotime, China) was used to detect cell necrosis rate in such study. In the PI/HO staining experiment, HT22 cells were seeded in the 24-wells-plates at a density of 1×10 5 / mL. After overnight incubated, pretreated with the indicated concentration of PPF for 2 h before erastin adding. After 8 h treatments of erastin, cells were incubated with 5 μg/mL PI and 10 μg/mL Hoechst 33342 for 15 min. The results were captured by Olympus uorescence microscope. Cell necrosis rate was calculated by PI (+)/Hoechst (+).