Genios pro

Intracellular lipid accumulation was determined enzymatically using the serum triglyceride determination kit (Sigma-Aldrich, St. Louis, MO, USA). Therefore, spheroid-laden hydrogels were harvested in 0.5% aqueous Thesit solution (0.5% Thesit in H₂O; Gepepharm, Hennef, Germany) and homogenized with a TissueLyser II (Qiagen, Hilden, Germany) at 25 Hz for 5 min, followed by sonification (Sonopuls; Bandelin electronic, Berlin, Germany). Subsequently, quantification of triglyceride content was carried out according to the manufacturer’s instructions and measured with a spectrofluorometer (Tecan GENios pro; Tecan, Crailsheim, Germany) at 570 nm. Results were normalized to the total amount of DNA in the respective hydrogel lysates. For quantification of the DNA content, hydrogels were harvested in phosphate/saline buffer (50 mM phosphate buffer, 2 mM Na₂EDTA * 2 H₂O, 2 M NaCl, pH 7.4; all obtained from Carl Roth, Karlsruhe, Germany) and homogenized as described above. Using the intercalating dye Hoechst 33258 (Polysciences, Warrington, USA), DNA content was determined fluorometrically (Tecan GENios pro; Tecan, Crailsheim, Germany) at an excitation wavelength of 365 nm and an emission wavelength of 458 nm. Salmon sperm DNA served as a standard.

Accumulation of intracellular lipids in differentiated ASCs was assessed as described in [28 (link)]. In brief, an analysis of triglyceride content was carried out using the triglyceride determination kit (Sigma-Aldrich). Cells were harvested in 0.5% aqueous Thesit solution (0.5% Thesit in H₂O; Gepepharm, Hennef, Germany) and subsequently sonicated (Sonopuls; Bandelin electronic, Berlin, Germany). Triglyceride content was quantified according to the manufacturer’s instructions with a spectrofluorometer (Tecan GENios pro; Tecan, Crailsheim, Germany) at 570 nm. To quantify the DNA content of the samples, cells were suspended in phosphate/saline buffer (50 mM phosphate buffer, 2 mM Na₂EDTA × 2 H₂O, 2 M NaCl, pH 7.4; all purchased from Carl Roth, Karlsruhe, Germany) and sonicated. DNA-intercalating dye Hoechst 33258 (Polysciences, Warrington, PA, USA) was used to determine DNA content fluorometrically (Tecan GENios pro; Tecan) at an excitation wavelength of 365 nm and an emission wavelength of 458 nm. Salmon sperm DNA served as a standard. Intracellular TG contents were normalized to the total amount of DNA in the corresponding sample.

Cells were stimulated with either GL or tert-butyl hydroperoxide (TBHP) (0.4 mM). After 3 h of incubation, cells were washed with PBS and incubated with 1 μM of 2,7-dichlorofluorescein diacetate (H2DCF-DA; Molecular Probes, OR, USA) for 20 min at 37°C in darkness. Fluorescence was measured at 450 nm excitation and 535 nm emission using a TECAN GENios Pro (Tecan Group Ltd, Switzerland).

As in the most of similar studies, the rate of ROS generation in mitochondria was estimated in state 2. Therefore, the rate of ROS generation was determined in state 2 using the incubation medium (IM) without creatine by a previously described method [33 (link)]. The rate of ROS generation was evaluated by an accumulated ROS amount after 30 min incubation at different temperatures ranging from 37 to 45 °C. After the incubation of 0.5 mg mitochondrial protein/mL with P+M or G+M, 5 μM Amplex Red (N-acetyl-3,7-dihydroxyphenoxazine) and 10 U/mL horseradish peroxidase, ROS amount was determined fluorimetrically using a thermostated fluorimeter “Tecan GENios Pro™” (Tecan Group Ltd., Menedorf, Switzerland). Measurements were carried out in 96-well plates at 535/590 ± 10 nm excitation/emission wavelengths. Calibration was performed by adding 5 μmol H₂O₂.

Three different colonies from the agar medium were grown in SDB. The cell concentrations were standardised using 0.5 M MacFarland solution to 2 × 10⁸ c.f.u/ml according to protocol by Ramirez-Arcos [19 (link)]. The broth microdilution assay was conducted to determine the effect of the two crude extracts and pure tormentic acid on both C. albicans and C. tropicalis according to Wiegand et al. [20 (link)]. Double dilution concentrations of 0 μg/ml to 100 μg/ml of the extract and tormentic acid were prepared in SDB. Fluconazole or miconazole was used as the positive control. Wells containing either the extract only or SDB only served as controls. The 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was done to investigate the presence/absence of viable cells in all wells after incubation according to [21 ]. A volume of 20 μl of the MTT solution was added to each of the 96 wells on the microtitre plates. The plates were incubated at 37°C for at least 2 hours. Color changes were quantified at 590 nm using a microplate reader (Genios Pro, Tecan Group Ltd., Grödig, Austria). The Minimum inhibitory concentrations (MIC) for the extract or tormentic acid were recorded. Graphs were plotted to show cell viability using GraphPad Prism6 (Version 6.0 GraphPad Software Inc., San Diego, California, USA).