Transmission Electron Microscopy of Cell Spheres

Spheres from PANC-1 and PK-1 cells were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), then postfixed for 1 h with 2% OsO₄ dissolved in distilled water. Cells were then dehydrated using an ethanol gradient, and embedded in Epon. Ultrathin sections were generated using an ultramicrotome and stained with uranyl acetate and lead citrate, as described previously, for examination under a transmission electron microscope (H-7500; Hitachi High-Technologies, Tokyo, Japan)^{17 (link),27 (link)}. Sphere formation was performed in triplicate for observation using TEM. To observe adherent cells using TEM, we cultured PANC-1 and PK-1 cells on cover slips in 24 well plates. The cells were fixed with 2.5% glutaraldehyde and then post-fixed for 30 min with 1% OsO_4. After dehydration using graded ethanol, Epon in capsules was placed on the cover slip. Three days later, the cover slip was heated at 100 °C on a hot plate, and then the cells on the cover slip were attached to the Epon. Subsequent procedures were performed as described above.