Fluorescent phallotoxins

Phase-contrast microscopic images of unstrained and strained hMSCs were obtained (Olympus, Japan) in at least four randomly selected sites from our visual field. To observe the effect of cyclic loading on cytoskeletal actin arrangements, hMSCs at all conditions were stained with fluorescent phallotoxins (Molecular Probes, Oregon, USA) for 30 min and then the nucleus stained with Hoechst (Molecular Probes, Oregon, USA) for 10 min in the dark. Fluorescence was recorded using a laser scanning confocal attachment (Leica TCS SP5 II, Germany) and measured by LAS AF image software (Leica, Germany). Images of unstrained MSCs on silicone membrane served as control.

For the detection of fragmented DNA, due to apoptosis at the cellular level in HCECs, TUNEL assay was performed using the APO-BrdU TUNEL assay kit (catalog number: A23210: Molecular Probes), according to the manufacturer’s protocol. HCECs were seeded at a density of 6 × 10⁴ cells per milliliter and grown on Nunc Lab-Tek II chamber slides (Thermo Fisher Scientific; Waltham, MA, USA) and 0, 0.125, 0.25, and 0.5 mg/mL of MXF were treated for 24 h. The cells were fixed with 3.7% paraformaldehyde for 10 min at room temperature, and permeabilization was carried out using 0.1% triton x-100 for 5 min at room temperature. Following the washing of steps with DPBS, the cells were blocked using 1% BSA (Sigma–Aldrich) in DPBS for 30 min at RT. The chamber slides were incubated overnight at 4 °C, which were labeled using TdT enzyme and anti-BrdU mixture solution. For the final detection of the broken site of DNA, Alexa Fluor 488 dye was labeled with incorporated anti-BrdU. The staining of F-actin was carried out using fluorescent phallotoxins (1 unit; catalog number: MP00354; Molecular Probes), and the counterstaining of cell nuclei was performed using 4′,6-diamidino-2′-phenylindole (DAPI, catalog number: 10236276001; Roche Diagnostics GmbH, Mannheim, Germany). The slides were viewed using a confocal laser scanning microscope LSM800 (Carl Zeiss, Oberkochen, Germany).