A Protocol for Quantifying Lipid Peroxidation in Cellular Systems by F2-Isoprostane Analysis
Figure 4
Characteristics of F2-isoprostane analysis in cells.
(A) 8-isoPGF2α-III and 5-iPF2α-VI quantification is not affected by ion suppression. UPLC-MS/MS intensities of increasing concentrations of 8-isoPGF2α-III and 5-iPF2α-VI standards in PBS or cell lysates relative to a fixed amount (2ng) of their respective deuterated internal standard were plotted. (B) F2-isoprostanes are stable in physiological conditions. 8-isoPGF2α-III-d4 and 5-iPF2α-VI-d11 were spiked into a HepG2 cell lysate and incubated at 25 or 37°C for indicated time points. F2-isoprostanes were measured as in Figure 4A and scatter plots show individual data points. (C-F) 8-isoPGF2α-III and 5-iPF2α-VI are excreted in the culture media at different rates by HepG2 cells. HepG2 cells were treated with GOX for 2 hours to induce oxidative damage where after GOX was removed and cells were allowed to recover for indicated times. F2-isoprostane levels were monitored as in Figure 4A. Results are shown as mean fold increase ± S.D (* p < 0.05, ** p < 0.01,).