High content analysis of differentiation and cell death in human adipocytes

Abstract

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...

SGBS cells as a model of human adipocyte browning: A comprehensive comparative study with primary human white subcutaneous adipocytes

“…This cell strain thus serves as an almost unlimited source of human preadipocytes and a widely used tool to study human adipocyte biology 12–17 .…”

Quantification of alignment of vascular smooth muscle cells

“…During the preprocessing, the data of the F‐actin labeled fluorescence images were adjusted, improving the contrast of the actin fibers: we used a generalization of the Otsu method to eliminate the image background noise, which is widely used for this purpose . That method establishes an optimum threshold , however, often, due the experimental setup, the noise and the signal are too close and the threshold may not be the best choice.…”

Measurement of Autophagy by Flow Cytometry

“…The mechanism of retention within the granules is not established although its fluorescence is not reversed by weak basic compounds. The use of LysoTracker probes have also been used to investigate the degree of autophagy occurring by measurement of their fluorescence by microscopy and to a limited extent by flow cytometry (Boya et al, 2003(Boya et al, , 2005Rodriguez-Enriquez et al, 2006;Byun et al, 2009;Mellen et al, 2009;Chikte et al, 2013). The new autophagy dye from Enzo, Lyso-ID has also been similarly used to monitor autophagy being co-localized with LC3-II during the autophagic process by image cytometry (Phadwal et al, 2012).…”

“…The biological detection and measurement of autophagy initially relied upon electron microscopy (Ashford and Porter, 1962;Deter and Duve, 1967) and more recently, timeconsuming image analysis and immunoblot techniques (Kabeya et al, 2000;Barth et al, 2010;Hansen and Johansen, 2011). Only recently has flow cytometry been employed to study the autophagic processes, which not only centers upon the detection, but also the semi-quantification of the autophagic biological marker, microtubule-associated protein LC3-II located in the autophagosome (Thomas et al, 2011;Phadwal et al, 2012;Chikte et al, 2013). This double-membrane-bound vesicle, the autophagosome, is the precursor to the energy-producing autolysosome, which is formed by its fusion with lysosomes (Tooze and Yoshimori, 2010;Yang and Klionsky, 2010).…”

“…Here we describe an antibody intracellular staining protocol for the semi-quantification of LC3-II during the process of autophagy by comparison of median fluorescent intensities (MFI) of autophagic cells induced by numerous inducers of autophagy, e.g., rapamycin, chloroquine and serum starvation,and compare this to that detected in untreated cells (Phadwal et al, 2012;Chikte et al, 2013). The generation of lysosomes during the autophagic process can be measured by the use of LysoTracker dyes, which detect lysosomal mass and whose fluorescent signal increases during autophagy (Boya et al, 2005;Rodriguez-Enriquez et al, 2006;Chikte et al, 2013). These two protocols are compared and their disadvantages are discussed.…”