Understanding adipocyte biology and its homeostasis is in the focus of current obesity research. We aimed to introduce a high-content analysis procedure for directly visualizing and quantifying adipogenesis and adipoapoptosis by laser scanning cytometry (LSC) in a large population of cell. Slide-based image cytometry and image processing algorithms were used and optimized for high-throughput analysis of differentiating cells and apoptotic processes in cell culture at high confluence. Both preadipocytes and adipocytes were simultaneously scrutinized for lipid accumulation, texture properties, nuclear condensation, and DNA fragmentation. Adipocyte commitment was found after incubation in adipogenic medium for 3 days identified by lipid droplet formation and increased light absorption, while terminal differentiation of adipocytes occurred throughout day 9-14 with characteristic nuclear shrinkage, eccentric nuclei localization, chromatin condensation, and massive lipid deposition. Preadipocytes were shown to be more prone to tumor necrosis factor alpha (TNFa)-induced apoptosis compared to mature adipocytes. Importantly, spontaneous DNA fragmentation was observed at early stage when adipocyte commitment occurs. This DNA damage was independent from either spontaneous or induced apoptosis and probably was part of the differentiation program. V C 2013 International Society for Advancement of Cytometry Key terms adipocyte; preadipocyte; SGBS; differentiation; DNA damage; laser scanning cytometry; high content analysis OBESITY is major risk factor for a number of chronic diseases, including diabetes, cardiovascular diseases, and cancer. In physiological context, body fat mass is driven via the balance between the adipocyte's growth and removal. Adipose tissue can grow by either hyperplasia or hypertrophy or both, whereas adipocyte removal is facilitated by apoptosis. Adipogenesis, when adipocytes develop from mesenchymal stem cells, is a fairly well known process (1-3). In current studies, multiparametric model systems have been developed, where adipocyte differentiation, its nuclear, cytoskeletal, and suborganellar morphological changes can be monitored together with different drug applications in an automated manner. Among these works, Lee et al. (4) had introduced a noticeable flow cytometric assay to assess differentiation of preadipocytes, based on cytoplasmic granularity changes corresponding to lipid droplet accumulation. However, application of flow cytometry, which requires cell suspensions, strongly violates physiological optima of solid tissues. This technique also omits the possibility of in situ visualization and reanalysis of samples attached to culture dish. These latter obstacles were solved by applying image cytometry in solid tissue quantifications, as in Lin's work, where laser scanning cytometry was used to study adipocytes and measure capacity of drugs to induce adipocyte apoptosis (5,6). Yet, cell recognition of attached cells was not solved or at least not clearly explained whether single cell conto...