Viable cells were quantified by trypan blue exclusion, as described elsewhere (Abecasis et al., 2017 (link)). For viability assessment, the enzyme substrate fluorescein diacetate (FDA, Sigma–Aldrich) and the DNA dye propidium iodide (PI, Sigma–Aldrich) were used (Serra et al., 2011 (link); Silva et al., 2015 (link)). In this method, direct staining of the live aggregates was performed followed by observation at the fluorescence microscope (DMI6000, Leica, Wetzlar, Germany), as described elsewhere (Serra et al., 2011 (link)). Cells that accumulated the metabolized product of FDA were considered live, and cells stained with PI were considered dead.
For evaluation of metabolic activity, the reduction capacity of the cultures was measured by PrestoBlue® Viability Reagent reduction assay (Life Technologies), according to the manufacturer’s instruction. PrestoBlue® reagent is reduced by viable cells and becomes highly fluorescent. This color change can be detected using fluorescence measurements. Samples of 2 to 5 capsules were taken from suspension agitated cultures into a 96-well plate. The microencapsulated cells were incubated with PrestoBlue® reagent for 3 h at 37°C. After this period, the supernatant was collected, and the fluorescence was read at 560-nm excitation and 590-nm emission in the microplate reader Infinite®200 PRO (NanoQuant, Tecan Trading AG).
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