Cell preparation for transmission electron microscopy (TEM)

Z310 cells were washed three times with PBS followed by digestion with 0.25% trypsin-EDTA for 4 min. Cells were collected into microcentrifuge tubes, followed by centrifugation (350 g for 10 min). Cell pellets were fixed with 3% GA for 3 h at room temperature. The samples were fixed with 0.5% osmium tetroxide in 0.1 mol/L PB for 1 h, dehydrated in graded series of ethanol, then in propylene oxide, and finally flat-embedded in Epon 812. Ultrathin sections (100 nm) were cut with the ultramicrotome and mounted on nickel mesh grids (6–8 sections/grid). Hitachi electron microscopy (H-7650; Hitachi Ltd, Tokyo, Japan) was used for observation of sections and EDS that can be used to calculate the constituent ratio including weight percent (Wt%) and atom percent (At%) of metallic elements.