Las af v 3 1 0 build 8587

UC MSCs seeded samples were treated with Hanks’ Balanced Salt solution (Merck, Darmstadt, Germany), fixed by 4% paraformaldehyde (5 min) and by cold methanol (1 min), and stained with antibodies ab15580 (Abcam, Cambridge, UK) against proliferation marker Ki-67 according to manufacturer’s recommendations. The second type of antibodies was PE-conjugated sc3739 (Santa Cruz Biotechnology, Dallas, TX, USA), cell nuclei were stained with DAPI (Merck, Darmstadt, Germany). The observations were carried out with the use of a Leica DM 4000 B fluorescent microscope and LAS AF v.3.1.0 build 8587 software (Leica Microsystems GmbH, Wetzlar, Germany).

For cell visualization and cell count on the surface of polymer films, UC-MSCs were labeled with fluorescent red-orange vital dye PKH26 (Merck, Darmstadt, Germany) before seeding the scaffolds according to the manufacturer’s protocol. This protocol uses proprietary membrane labeling technology to incorporate a yellow-orange fluorescent dye with long aliphatic tails (PKH26) into lipid regions of the cell membrane [62 (link)]. The appearance of labeled cells may vary from bright and uniform to punctate or patchy, depending on the cell type being labeled and the extent to which membrane internalization occurs after labeling [63 (link)]. Cell nuclei were counterstained by Hoechst 33,342 (5 mg·mL⁻¹, ThermoFisher Scientific, Waltham, MA, USA) within 10 min.
The observations were carried out with the use of a Leica DM 4000 B fluorescent microscope and LAS AF v.3.1.0 build 8587 software (Leica Microsystems GmbH, Wetzlar, Germany).