A simplified workflow with end-point validation of real-time electrical cell-substrate impedance sensing of retinoic acid stimulated neurogenesis in human SH-SY5Y cells in vitro.

OBJECTIVE: Retinoic acid (RA) is known to transition proliferating SH-SY5Y neuroblastoma cells towards functional neurons. However, the activity of RA is restricted due to its photolability where any findings from prolonged time course observations using microscopy may alter outcomes. The aim of the study was to establish a real-time, long-term (9-day) protocol for the screening of differentiation events using Electrical cell-substrate impedance sensing (ECIS). RESULTS AND DISCUSSION: A differentiation baseline for SH-SY5Y cells was established. Cells were seeded and exposed to repeated spikes of RA using the xCELLigence real-time cell analyser single plate (RTCA-SP) for real-time monitoring and identification of differentiation activity over a 9 day period in order to be more representative of differentiation over a prolonged timeline. Specific features associated with differentiation (growth inhibition, neurite outgrowths) were confirmed by end-point analysis. RA-induced growth inhibition and assumed phenotypic changes (i.e. neurite outgrowth) were identified by the xCELLigence analysis and further confirmed by end-point metabolic and phenotypic assays. Change in cellular morphology and neurite outgrowth length was identified by end-point fluorescence detection followed by computational analysis. Based on this it was possible to identify SH-SY5Y phenotypic differentiation with distinct phases observed over 9 days using Electric cell-substrate impedance sensing (ECIS) cell index traces providing a path to application in larger scale neurotrophic factor screening using this scalable technology.

Real-time monitoring of 3T3-L1 preadipocyte differentiation using a commercially available electric cell-substrate impedance sensor system.

Real-time analysis offers multiple benefits over traditional end point assays. Here, we present a method of monitoring the optimisation of the growth and differentiation of murine 3T3-L1 preadipocytes to adipocytes using the commercially available ACEA xCELLigence Real-Time Cell Analyser Single Plate (RTCA SP) system. Our findings indicate that the ACEA xCELLigence RTCA SP can reproducibly monitor the primary morphological changes in pre- and post-confluent 3T3-L1 fibroblasts induced to differentiate using insulin, dexamethasone, 3-isobutyl-1-methylxanthine and rosiglitazone; and may be a viable primary method of screening compounds for adipogenic factors.

Guardian of the furnace: mitochondria, TRAP1, ROS and stem cell maintenance.

Mitochondria are key to eukaryotic cell survival and their activity is linked to generation of reactive oxygen species (ROS) which in turn acts as both an intracellular signal and an effective executioner of cells with regards to cellular senescence. The mitochondrial molecular chaperone tumor necrosis factor receptor associated protein 1 (TRAP1) is often termed the cytoprotective chaperone for its role in cancer cell survival and protection from apoptosis. Here, we hypothesize that TRAP1 serves to modulate mitochondrial activity in stem cell maintenance, survival and differentiation.