12 well plate

Cells were plated on 18 mm coverslips in a 12-well plate (Warner Instruments Corporation, Hamden, CT, USA) at a density of 250,000 cells in 1 mL of complete medium for microscopy experiments and on 6-well plates at a density of 500,000 cells per well in 2 mL of complete medium for flow cytometry experiments. Cells were allowed to recover for 24 h and then were exposed to AgNPs or Ag⁺ for 24 h at 37 °C. Medium was removed and fresh media containing 10 µM of the lipid peroxide specific dye, Liperfluo (Dojindo Molecular Technologies, Rockville, MD,) was added for 30 min. Cells were then washed twice with PBS and fluorescence was measured using an Olympus FV1200 spectral laser scanning confocal microscope and a FACS Canto II Analyzer (BD Biosciences). Analysis of the data was performed using FCS express version 7 (De Novo Software, Glendale, CA, USA).

Cells were plated on 18 mm coverslips in a 12-well plate (Warner Instruments Corporation, Hamden, CT, USA) at a density of 250,000 cells in 1 mL of complete medium. Cells were allowed to recover for 72 h and then were exposed to AgNPs or Ag⁺ for 24 h at 37 C. Medium was removed and cells were fixed with 4% formaldehyde solution and permeabilized (0.5% Triton X-100, 3 mm EDTA, pH 8.0). Cells were then stained with Proteostat Aggresome Detection Reagent (1:1000) and Hoechst 33,342 (1:1000) (Enzo Biosciences, Ann Arbor, MI) diluted in 1X PBS for 30 min, washed twice with PBS, and coverslips were mounted on glass slides with Prolong Gold antifade reagent (Invitrogen). Fluorescence was visualized using an Olympus FV1200 spectral laser scanning confocal microscope.