Cyquant xtt assay

Mitochondrial activity was determined with the CyQUANT™ XTT assay (Thermo Fisher, Waltham, MA, USA). A mixture of XTT reagent and electron coupling reagent was prepared at a ratio of 6 to 1. Seventy microliters of the mixture were added to each well. Then, the plate was incubated at 37 °C for 4 h in the dark in a 5% CO₂ incubator. The optical densities (OD) were determined using the SpectraMax^® Plus 384 Microplate Reader (SpectraMax M3; Molecular Devices, SanJosé, CA, USA) at 450 nm and 660 nm [31 (link),34 (link)]. Mitochondrial activity was expressed by the decrease in OD (450–660 nm), discounting the average of the ODs of the controls [35 (link)]. The isolates were categorized into three groups based on their metabolic activity: low metabolic activity, moderate metabolic activity, and high metabolic activity. The classification was determined using XTT with respective cut-offs of <0.097, 0.097–0.2, and >0.2 [33 (link)].

Healthy cat claws were crushed with a mortar and pestle, sterilized by autoclavation, and subsequently mixed with a minimum medium (MM) containing 15 mM glucose, 10 mM MgSO₄7·H₂O, 29 mM KH₂PO₄, 13 mM glycine, and 3 µM thiamine (all compounds from Merck Millipore, Darmstadt, Germany) to form a suspension of 2% w/v powdered claws. An inoculum of 1 × 10⁶ yeast cells/mL was prepared in MM with or without (control) powdered claws and added to the wells of a 96-well plate in a 200 μL/well volume. Plates were incubated at 35 °C for 7 days, and the growth was analyzed by the absorbance measurement at 530 nm using a microplate reader (SpectraMax M3; Molecular Devices, San Jose, CA, USA). Mitochondrial activity was determined with the CyQUANT XTT assay (Thermo Fisher, Waltham, MA, USA). A mixture of XTT reagent and electron coupling reagent was prepared at a ratio of 6 to 1. Seventy microliters of the mixture were added to each well. Then, the plate was incubated at 37 °C for 4 h in the dark in a 5% CO₂ incubator. The optical densities (ODs) were determined, in two technical replicates, using the SpectraMax^® Plus 384 Microplate Reader (SpectraMax M3; Molecular Devices, San Jose, CA, USA) at 450 nm and 660 nm. Mitochondrial activity was expressed by the decrease in OD (450–660 nm), discounting the average of the ODs of the controls [29 (link)].