Xtt assay

Cytotoxicity was evaluated in HeLa, PC12 [54 (link)], originally purchased from Clontech Laboratories Inc., Cat. No. 630912) and SH-SY5Y cells [17 (link)] after 3 days of incubation with the test compounds by using a tetrazolium dye assay (XTT assay, AppliChem GmbH, Darmstadt, Germany).

The inhibitors were added to the cultured cells from stock solutions in DMSO to the desired final concentration. For cytotoxicity assays, HeLa cells were grown in 96-well plates (20,000–30,000 cells per well) and incubated with the test compounds for 3 days before cell viability was evaluated with the help of a tetrazolium dye assay (XTT assay, AppliChem GmbH, Darmstadt, Germany).
Inhibitor assays of endogenous DYRK1A activity were performed with overexpressed GFP-SF3B1-NT as described previously [21 (link)]. Briefly, HeLa cells were grown in 6-well plates and treated with test compounds for 18 h before cells were lysed with 100 μL SDS lysis buffer (20 mM Tris HCl pH 7.4, 1% SDS) at 96 °C and sonicated. Total cellular lysates were analysed by immunoblotting with a custom-made rabbit antibody for detecting phosphorylated T434 [43 (link)] and a goat antibody for GFP (no. 600-101-215, Rockland Immunochemicals, Gilbertsville, PA, USA). pT434 signals were quantified using the AIDA Image Analyzer 5.0 program (Raytest, Straubenhardt, Germany). Relative phosphorylation of SF3B1 was calculated by normalisation to total protein levels as determined from GFP immunoreactivity. IC₅₀ values were determined by non-linear curve fitting using the GraphPad Prism 5.0 program (GraphPad Software, La Jolla, CA, USA).

The impact of HL EVs isolated from KM-H2 cell culture on the proliferation of HDF_n cells and vice versa was probed using the XTT assay (AppliChem). 6 × 10³ KM-H2 or 1 × 10⁵ HDF_n cells per well were seeded on a 96-well plate. Cells were incubated with the amount of EVs and time period indicated in the according figure. The XTT staining solution was prepared according to the manufacturer’s protocol, 50 µl staining solution added to each well containing 100 µl growth medium, and incubated for 2 h at 37°C. Absorbance was then measured with an Infinite M1000 microplate reader (Tecan) at a wave length of 475 and 660 nm as reference.

To determine the viability of cells treated with UCB-MNC-sEV, an XTT assay (Applichem) was performed according to the supplier’s instructions. Briefly, the XTT mix was added to the medium and incubated at 37 °C for 3.5 h. The absorbance was read at 450 nm and 630 nm.

Metabolic activity of mature HepaRG cells was determined using the XTT assay according to the manufacturer’s instructions (AppliChem). Briefly, XTT reagent (1 mg/mL) was added on cell-laden 3D constructs and incubated for 4 h (37 °C, 5% CO₂). The absorbance of the supernatant was measured spectrophotometrically at A450 nm (TriStar Multimode Reader LB942, Berthold Technologies, Bad Wildbad, Germany) with a reference of A620 nm.
Lactate dehydrogenase (LDH) release of mature HepaRG cells was measured with the LDH detection kit (Roche, Grenzach, Germany), according to the manufacturer’s instructions, in the supernatant at an absorbance of A492 nm with a reference of A620 nm (Sunrise absorbance microplate reader, Tecan). XTT and LDH values were normalized to lysis controls. For cell lysis, cell-laden 3D constructs were incubated in culture medium supplemented with 10% Triton-X-100.
The LIVE/DEAD assay of mature HepaRG cells was performed with the Viability/Cytotoxicity kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Briefly, cell-laden 3D constructs were stained with 2 µM calcein-AM and 2 µM ethidium homodimer-1 diluted in 1x Hank’s balanced salt solution (HBSS; Thermo Fisher Scientific) for 30 min (37 °C, 5% CO₂). The stained cell-laden 3D constructs were analyzed by fluorescence microscopy (Zeiss Observer. Z1 microscope).