Infinite m nano microplate reader

The fibronectin adhesion assay was performed in 96-well flat-bottom plates as previously described^{77 (link)}, with minor changes. Briefly, the wells were coated with 200 μl of human fibronectin (Corning, 356008) at 50 μg/ml (18 h, 4 °C). Wells were then washed three times with PBS supplemented with 1% FBS (20 min, 37 °C). Overnight S. aureus cultures were adjusted to OD₆₂₀ 1.0, corresponding to ca. 1 × 10⁹ CFUs/ml. One ml of each bacterial suspension was centrifuged (12,000 × g, 2 min), and pellets were washed and resuspended in PBS. 100 μl of each bacterial suspension were added to the wells of the fibronectin-coated plate and incubated for 30 min at 37 °C with mild shaking. Wells were washed three times with PBS to remove non-adherent bacteria. Adherent bacteria were fixed with glutaraldehyde (2.5% v/v in 0.1 M phosphate buffer for 2 h at 4 °C) and stained with crystal violet (0.1% m/v; Elitech, SS-041C-EU) for 30 min at room temperature. After three washes with PBS, the total remaining stain impregnating the adherent bacteria was solubilized using 100 μl of 0.2% Triton X-100 at room temperature for 30 min. Quantification of adherent bacteria was performed by measuring OD₅₉₀ using an Infinite M Nano+ microplate reader (Tecan). The values were normalized to the reference strain S. aureus 8325-4; S. aureus DU5883 was used as a control.

RNA isolation from skin cells, followed by cDNA synthesis, was performed as described previously [30 (link), 31 (link)]. Briefly, cells were lysed with TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer's instructions, and total RNA was extracted. The yield and purity of RNA were determined using the Infinite M Nano microplate reader (Tecan). Then, 1 μg of total RNA from each sample was reversely transcribed using GoScript™ Reverse Transcription Mix, Oligo(dT) (Promega, WI, USA), according to the manufacturer's instructions.
RNA isolation from dermal equivalents, followed by cDNA synthesis, was carried out as described previously [32 (link)]. In brief, total RNA was extracted from Aronia extract-treated tissues using RNeasy Mini Kit (Qiagen, Germany). The yield and purity of the isolated total RNA were measured using the NanoDrop® spectrophotometer (Thermo Fisher Scientific). Then, 1 μg of each total RNA sample was reversely transcribed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA), according to the manufacturer's instructions.

For cell culture and maintenance, normal African green monkey kidney fibroblast cells (Vero; ATCC^® CCL-81™) and immortalized human keratinocyte cell line (HaCaT; CLS 300493) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Invitrogen, Carlsbad, CA, USA), supplemented with 10 % fetal bovine serum (v/v), and 1% Antibiotic-antimycotic (v/v) (Gibco, Invitrogen, CA, USA) at 37 °C in the present of 5% CO_2.To test the cytotoxicity effects of normal cell cultures, Vero and HaCaT cells were seeded approximately 1.5 × 104 cells per well into 96 well-plates (Falcon^® a Corning brand, USA) and incubated for 24 h. All cells were then treated with 0 (distilled water only), 500, 1000, and 1500 μg /mL of crude extract. After 96 h of incubation, the mixing solutions were discarded; then, 4 μg of MTT solution was added and incubated for 3 h. Lastly, MTT solution (Invitrogen, USA) was replaced with 50 μL of dimethyl sulfoxide (Thermo Fisher Scientific, CA, USA) prior to the absorbance measurement at 570 nm by using a Infinite^® M Nano microplate reader (Tecan, Männedorf, Switzerland).