Ultrastructural Analysis of CuCl2-Treated Cells

Electron microscopy was performed as previously described (10 (link)). Briefly, MN9D cells grown in petri dishes were treated with 250 μM CuCl₂ for 15 h followed by fixation with a mixture of 2% formaldehyde and 0.2% glutaraldehyde (Polysciences, Inc, 01,909) in 0.1 M cacodylate buffer (pH 7.2) for 30 min at 37 °C. To stop the fixation step, free aldehyde groups were blocked by soaking cells in 50 mM ammonium chloride in 0.1 M cacodylate buffer for 1 h. Cells were removed, sedimented by centrifugation, enclosed in liquefied 2% agarose, and post-fixed for 1 h with 1% osmium tetroxide (Electron Microscopy Sciences, EMS, 19,152). Subsequently, en bloc staining was performed with 1% aqueous uranyl acetate for 1 h. After staining, cells were dehydrated using an ethanol series and embedded in epoxy resin (Fluka, 45,345). Ultrathin sections (80 nm) were prepared, placed on Cu slot grids, stained with uranyl acetate and lead citrate, and observed at 80 kV with a Hitachi H-7650 electron microscope (Hitachi). Electron micrographs were taken with an 11-megapixel CCD XR611-M digital camera (Advanced Microscopy Techniques).