A Simple Method for Classification of Cell Death by Use of Thin Layer Collagen Gel for the Detection of Apoptosis and/or Necrosis after Cancer Chemotherapy

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Abstract
To assess the efficacy of cancer chemotherapy, an important index is apoptosis of the target cells, which can usually be confirmed by electron microscopy (EM). We established a new experimental technique, whereby cancer cells (MKN45) were distributed in thin collagen gel as one or two cell layers, and cultured with anti‐cancer drugs (5‐FU and CDDP). The cells were stained with fluorescent Hoechst 33258 (Ho) and photographed, then with hematoxylin and eosin (H&E) and again photographed, and processed for EM. This approach allowed us to characterize the patterns of death of single cells in detail. There were six patterns of cell damage: two patterns of apoptosis, early peripheral condensation of chromatin and late apoptotic bodies, two patterns of necrosis, cytoplasmic swelling and washed‐out images, and two further patterns, with morphological features of both apoptosis and necrosis, neither classified into necrosis nor apoptosis. The results show that cell death patterns can be mostly determined by combining observations of Ho and H&E‐stained cells without the necessity for EM observation.