AUTOMATED FLUORESCENCE MICROSCOPIC MEASUREMENT OF APOPTOSIS FREQUENCYFOLLOWING IONIZING-RADIATION EXPOSURE IN CULTURED-MAMMALIAN-CELLS

Purpose: To develop and assess an automated image cytometric method of apoptotic cell classification for use under conditions in which apopt osis is a rare event (e.g, fibroblastoid cell lines or low-dose irradi ation). Methods: Image acquisition software was adapted to gather doub le-stained cell images from slides prepared using cell fixation and st aining methods that emphasized apoptotic morphology. Chinese hamster o vary cells (CHO) were classified individually by discriminant analysis of morphological and nuclear texture features calculated for each ima ge. Discriminant functions were constructed from a manually classified set of over 60 000 cell images categorized as 'normal', 'apoptotic', 'cell doublers' or 'debris' and all subsequent cell images collected w ere classified using these functions. Results: Application of this tec hnique resulted in a 99.8% accuracy in classification of the normal ce ll 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 unti l 18 h post-irradiation, which corresponded with the release of the G2 block as determined by DNA-content analysis. Apoptotic frequency incr eased to a peak level of 12.1 +/- 4.6% at 42 h post-irradiation. Concl usions: Automated image cytometry provides an efficient and consistent method of apoptosis measurement. This study represents the first deta iled characterization of the time course and the role of cell division in CHO cell apoptosis.