Resazurin

mOECs or Schwann cells were seeded at a density of 2000 cells per well in a 384-well microplate (Greiner). After 24 h, the culture media was removed and different treatments in complete media were added: (1) negative control: 0.2% DMSO, (2) positive control: 1% G5 supplement (100X), 0.2% DMSO, (3) concentrations from 0.02 to 12.5 µM of RAD288 and RAD289, 0.2% DMSO. To assess cell viability, after 24 h incubation with the different treatments, 5 µL of resazurin (500 µM, Sigma Aldrich) were added to each well leading to a resazurin final concentration of 50 µM. The plate was then incubated for 4 h at 37°C and 5% CO₂. The fluorescent signal was quantified with an EnVision™ Multilabel (Perkin Elmer) plate reader at 535/595 nm. Then the cells were fixed in 4% PFA for 10 min. After fixation, cells were washed 3 times with PBS and stained with Hoechst (1:5000, Life Technologies, New Zealand) for 10 min and then washed 3 times with PBS. Cells were imaged automatically using Operetta^™ (PerkinElmer), a high content imaging system using a 20X high numerical aperture objective lens. Individual cell segmentation and cell count were performed using the Harmony 3.5.2^® software.

To determine the metabolic cell activity, a resazurin assay was performed. Before usage, the resazurin stock solution (0.11 mg/mL resazurin (No. R7017, Sigma-Aldrich) in PBS+ was diluted with phenol red free medium (1:100). All medium was removed from the cells and replaced with 350 µL of the resazurin solution per well (48-well plate). After 4 h of incubation at 37 °C and 5% CO₂ in an incubator, triplicates of 100 µL of the metabolized resazurin solution were pipetted into a 96-well plate and measured at a SpectraMax^® iD3 microplate reader (absorbance: 570 nm, reference wavelength: 600 nm).

Keratinocytes were cultured in 12-well plates for 48 hrs in EpiLife medium supplemented with either 100 nM calcipotriol or DMSO and without antibiotics. C. albicans or P. aeruginosa (patient isolate) were grown to early log phase in Luria broth (LB). 35,000 colony forming units/well were seeded into a 96-well black microplate (Greiner Bio-One, Kremsmünster, Austria) and 100 µl of conditioned cell culture medium from untreated or treated cells were added. After 3 hrs incubation at 37 °C, 100 µl of resazurin solution (0.5 nM resazurin, Sigma-Aldrich) was added. Plates were incubated at 37 °C overnight in a Spark 10 M multiplate reader (Tecan, Grödig, Austria) which measured fluorescence at Ex 535 nm/Em 590 nm every hour. C. albicans and P. aeruginosa were treated directly with increasing concentrations of LL-37, 100 nM calcipotriol, or DMSO served as controls.

For both treatment strategies, the viability of the GC-1 cells was measured using the Resazurin reduction assay. Viable cells with an active metabolism contain coenzymes, such as NADH, which are used in diaphorases to reduce Resazurin (blue, non-fluorescent) to resorufin (pink, fluorescent) [37 (link)]. The level of reduction is proportional to the number of viable cells. Resazurin was chosen to perform a cell viability assay because it is not toxic and, therefore, cells exposed to it can remain in the culture to be used for other experimental purposes [38 (link)].
The cells were incubated with 10% of the culture volume with a stock solution of Resazurin (Sigma-Aldrich) (0.1 mg/mL) in 1x PBS [39 (link)] 4 h before the end of the 6 and 12 h incubation timepoints and 4 h before the end of the 4-day recovery period. After 4 h of incubation, 100 µL of the supernatant from each sample was transferred to a 96-well plate, and the absorbance of Resazurin was measured spectrophotometrically at 570 and 600 nm (Infinite M200, PRO, Tecan). All absorbance values were corrected against blank wells containing cell-free culture medium with ZnO NPs and different concentrations of chalcone 1. Cells were visualized daily, under an inverted light microscope (EVOS™ M5000 Imaging System), to confirm cell confluence and morphology.

The effect of 6-OHDA and rotenone toxicity was also evaluated by measuring the cellular metabolic activity using the resazurin reduction assay. The resazurin assay is based on the reduction of resazurin (Cat. R7017-5G, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) to resorufin by dehydrogenases present in viable cells [39 (link)]. After cell treatments, the medium was replaced by a fresh medium containing resazurin (10 μg/mL) and kept in a humidified atmosphere, 5% CO₂, at 37 °C for 3 h. The fluorescent signal was monitored using a 540 nm excitation wavelength and 590 nm emission wavelength in a Cytation 3 microplate reader. A blank condition (wells without cells) was used as a fluorescent reference signal and subtracted from the remaining conditions. Data were normalized to the average control (100%) condition.