Omega plate reader

Extracting total RNA was performed using theTriFAST reagent (Peqlab, Erlangen, Germany), according to the manufacturer’s instructions. RNA concentration was measured using spectrophotometer (Omega plate reader, BMG Labtech, Offenburg, Germany) and 1 µg of total RNA was reverse transcribed to generate the complementary DNA (cDNA) with the First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany). PCRs were performed using Biozym Ready Mix (Biozym, Hessisch Oldendorf, Germany). Primer sequences and PCR conditions optimized for each primer set are summarized in Table 1.Representative images from the optimization see Figure S1. GAPDH was used as internal control. To obtain PCR results in the linear range 10 ng of cDNA (with the exception of 20 ng for ACAN) was used as a template for each set of PCR. PCR products were separated by gel electrophoresis and visualized by ethidium bromide using the pUC19/Msp1 marker (Carl Roth, Karlsruhe, Germany) as a size reference. Densitometric analyses were performed to quantify signal intensities using the ImageJ software (NIH, Bethesda, MD, USA).

The specificity of KLK13 at nonprime subsites was evaluated using an ABZ/ANB library and the enzyme in concentrations ranging from 8.32 × 10⁻⁸ to 1.75 × 10⁻⁸ M. Deconvolution of the primed subsite library was carried out using KLK13 at concentrations from 1.25 × 10⁻⁸ to 1.25 × 10⁻⁹ M. Deconvolution was performed using the iterative method in solution [41 (link)]. Freeze-dried samples of each sub-library were dissolved in dimethyl sulfoxide (DMSO) to the final concentration of 5 mg/mL. 20 mL of each tested sub-library was supplemented with an assay buffer (180 μL; 50 mM Tris-Cl (pH 7.5) containing 1 mM EDTA and 0.5 μM heparin [20 (link)] and with the enzyme. All measurements were performed using the Omega plate reader (BMG Labtech, Ortenberg, Germany). Hydrolysis was monitored for 30 min at 37 °C by the ANB absorbance at 410 nm (for the non-primed library) or at ABZ excitation wavelength of 320 nm and emission at 450 nm (for the primed library).
The site of hydrolysis was determined after each step of deconvolution of primed site library and for both final substrates (1 and 2) by RP-HPLC monitored by fluorescence detection and MS.

Scaffolds containing cells were washed once with PBS and then incubated with 0.0025% resazurin in plain culture medium. Scaffolds without cells were used as background control. After incubation for 2 h at 37 °C and 5% CO₂ in humidified atmosphere the produced resorufin was measured by the fluorescence at 544 nm/590–10 nm using the Omega Plate Reader (BMG Labtech, Ortenberg, Germany) [40 (link)]. Therefore, 2 × 50 µL of each sample were transferred into cavities of fresh 96-well-plates.

To isolate the total DNA of cells differentiated on the scaffolds, samples were washed once with PBS with the help of a cell strainer and centrifugation (600 g, ambient temperature, 10 min). Scaffolds without cells were used as background control. Scaffolds were incubated in 400 µL of 50 mM NaOH for 30 min at 98 °C. Following incubation, samples were vortexed and frozen at −80 °C for at least 1 h. After thawing 400 µL of a 100 mM Tris buffer (pH = 8.0) was added to each sample to neutralize pH. These samples were centrifuged at 14.000 g, at 4 °C for 10 min before supernatants were transferred into fresh reaction tubes. DNA concentration was determined photometrically using the LVIS plate and the Omega Plate Reader (BMG Labtech) [41 (link)].