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. 2017 Nov 14;8(64):108006–108019. doi: 10.18632/oncotarget.22444

Figure 4. CD increases the generation of intracellular reactive oxygen species (ROS) to modulate cell death in NPC-BM and NPC-039 cells.

Figure 4

The amount of intracellular ROS was assessed using a DCFH-DA fluorescent probe and a flow cytometry. The green DCF fluorescence reflects intracellular ROS production. (A) Following treatment of cells with indicated concentrations of CD (0-8 μM) for 24 h, intracellular ROS generation was detected by flow cytometry using DCFH-DA staining. M1: negative control peak; M2: DCFH positive peak. (B) Percentage of green DCF fluorescence positive cells over total number of cells were calculated following 30, 60, 90, 120, 150, and 180 min exposure of 8 μM CD. (C-E) Cells were pretreated with or without 10 mM N-acetyl-L-cysteine (NAC; ROS scavenger) for 4 h, and then treated with or without 8 μM CD for 24 h. For quantitative analysis of apoptosis, cells were dual-labeled with PI and Annexin V fluorescence and analyzed by Muse Cell Analyzer flow cytometry (C). The scavenging ability of NAC for CD-induced ROS production was evaluated by percentage of green DCF fluorescence (D). Cell viability was determined by MTT assay (E). Data are representative of at least three independent experiments. Results are shown as mean ± SEM. *P < 0.05, compared with the control (0 μM). #P < 0.05, compared with cells treated with CD (8 μM).