| Autor: |
Golla N; The Hormel Institute, University of Minnesota, Austin, MN, USA., Hong LJ; Division of Gynecologic Oncology, Department of Gynecology and Obstetrics, Loma Linda University School of Medicine, Loma Linda, CA, USA., Chefetz I; The Hormel Institute, University of Minnesota, Austin, MN, USA. ichefetz@umn.edu.; Masonic Cancer Center, Minneapolis, MN, USA. ichefetz@umn.edu.; Stem Cell Institute, Minneapolis, MN, USA. ichefetz@umn.edu.; Department of Obstetrics, Gynecology and Women's Health, Minneapolis, MN, USA. ichefetz@umn.edu. |
| Abstrakt: |
Transmission electron microscopy (TEM) is an all-in-one tool to visualize the complex systems of any specimen that is 1 nm in size or smaller. The current chapter provides detailed guidelines for imaging morphological changes during programmed cell necrosis using TEM as a single-step methodology. In this protocol, a novel aldehyde dehydrogenase inhibitor is used to induce cell programmed necrosis in ovarian cancer cell lines (A2780 and SKOV3). This process is followed by gradient dehydration with ethanol, chemical fixation, sampled grid preparation, and staining with 0.75% uranyl formate. Following fixation and grid preparation, cells are imaged using TEM. The resulting images reveal morphological changes consistent with necrotic morphology, including swelling of cells and organelles, appearance of vacuoles, and plasma membrane rupture followed by leakage of cellular contents. The current approach allows a single-step methodology for characterization of cell-programmed necrosis in cells based on morphology. |
