Multicycle program

C6 cells (5 × 10⁵) were grown on 100-mm dishes for 24 h followed by treatment with vehicle (0.1% DMSO) or 20 nM (R,R ′)-MNF in serum-free medium for 6, 12, 24 and 48 h. Cell cycle distributions were performed by flow cytometry on propidium iodide-stained nuclei prepared by the NIM technique [26 ]. DNA histograms of at least 10,000 cells acquired on Becton-Dickinson FACScanto II (BD Biosciences) were deconvoluted using the Multicycle program (Phoenix Flow Systems) for estimates of the percentage of cells in the G0/G1, S, and G2+M phases of the cell cycle.

A549 cells were grown on 6-well plates to 70% confluence, washed once with warm PBS, and incubated with 5 μM 2′-7′-dichlorodihydrofluorescein diacetate (H₂DCFDA, Invitrogen) in PBS supplemented with 5.5 mM glucose. After 30 min at 37°C, PBS was replaced with complete culture medium and incubated for another 50 min at 37°C. Finally, cells were trypsinised and resuspended thoroughly with 0.4 mL of PBS, H₂DCFDA (50 μM) and PI (20 μg mL⁻¹). Intracellular internalised probe reacts with ROS and emits fluorescence when excited at 492 nm. Emitted fluorescence was recorded by flow cytometry at 520 nm using an Epics XL flow cytometer (Coulter Corporation, Hialeah, FL, USA). Data of DCF fluorescence concentrations from 1×10⁴ PI negative cells were collected and analysed using Multicycle program (Phoenix Flow Systems, San Diego, CA, USA).

5×10⁴ A549 cells/well were seeded in 6-well plates and treated 24 hours later with MSA for 24, 48 and 72 hours. Both adherent and detached cells were collected by centrifugation after trypsinisation, resuspended in 0.5 mL PBS and added dropwise to 4.5 mL 70% (v/v) cold ethanol. Cells were stained in PBS containing 50 mg mL⁻¹ propidium iodide (PI), 0.2 mg mL⁻¹ DNAse free RNAse (Roche, Basel, Switzerland) and 0.1% Triton X-100. Fluorescence-activated cell sorter (FACS) analysis was carried out at 488 nm in an Epics XL flow cytometer (Coulter Corporation, Hialeah, FL, USA). Data of 1×10⁴ cells were collected and analysed using Multicycle program (Phoenix Flow Systems, San Diego, CA, USA). All experiments were performed three times with three replicates per experiment.