Automated fluorescence microscopic measurement of apoptosis frequency following ionizing radiation exposure in cultured mammalian cells

Abstract

Purpose: To develop and assess an automated image cytometric method of apoptotic cell classification for use under conditions in which apoptosis is a rare event (e.g. fibroblastoid cell lines or low-dose irradiation). Methods : Image acquisition software was adapted to gather double-stained cell images from slides prepared using cell fixation and staining methods that emphasized apoptotic morphology. Chinese hamster ovary cells (CHO) were classified individually by discriminant analysis of morphological and nuclear texture features calculated for each image. Discriminant functions were constructed from a manually classified set of over 60 000 cell images categorized as 'normal', 'apoptotic', 'cell doublets' or 'debris' and all subsequent cell images collected were classified using these functions. Results : Application of this technique resulted in a 99.8% accuracy in classification of the normal cell population, and 81.7% classification accuracy for apoptotic cells. This method was then applied to study the time course of the apoptotic response of CHO cells following X-irradiation. Following irradiation with 5 Gy no increase above control levels of apoptosis was noted until 18 h post-irradiation, which corresponded with the release of the G2 block as determined by DNA-content analysis. Apoptotic frequency increased to a peak level of 12.1 4.6% at 42 h post-irradiation. Conclusions : Automated image cytometry provides an efficient and consistent method of apoptosis measurement. This study represents the first detailed characterization of the time course and the role of cell division in CHO cell apoptosis.