Dmi8 m system fluorescence microscope

Cells were seeded and cultured in 24-well culture plates at a density of 1 × 10⁵ cells per cm² and treated with iPA. For immunofluorescent staining, cells were fixed with 3.7% paraformaldehyde, permeabilized with 0.2% Triton X-100 and blocked using PBS-BSA 0.4%. Subsequently, cells were incubated with primary antibody at 4 °C overnight. The next day, cells were incubated with a labeled secondary antibody at room temperature for 1 h. DAPI (Hoechst, Life Technologies Corporation, Thermo Fisher Scientific, Waltham, MA, USA) in PBS was used for nuclei staining and rhodamine-phalloidin was used as the actin staining reagent (purchased from Abcam, Cambridge, UK). Finally, cells were mounted on slides using Dako Fluorescent Mounting Medium. The images were acquired using a Leica DMi8 M System fluorescence microscope.

Angiogenesis was measured using a tube formation assay. Cells were seeded in 96-well plates, pre-coated using 50 µL/well of Geltrex™ (Gibco) with ECM proteins concentration of 10 mg/mL. The coating was allowed to polymerize at 37 °C for 30 min. The cells were dissociated by trypsinization, washed in PBS (+Mg²⁺, +Ca²⁺), and re-suspended in serum-free medium at 3 × 10⁴ cells/mL. Subsequently, 0.1 mL/well cell suspensions were plated onto the surface of Geltrex™ and incubated at 37 °C for 24 h. Following incubation, each well was analyzed directly under a Leica DMi8 M System fluorescence microscope, tubes in each field were imaged and the number of branch sites/nodes (sites of intersection of least three tubes) was counted using the ImageJ.Ink software (1.51i, NIH, Bethesda, MD, USA). The average branch point number from 5–6 random fields in each well was reported.