Dmi6000 cs fluorescence microscope

Apoptosis of PASMCs was assessed using the Alexa Fluor 488 Click-iT™ Plus TUNEL Assay (ThermoFisher Scientific, Cat# C10617). Isolated PASMCs were cultured in 96-well plates (10,000 PASMCs/cm², passage 1) under hypoxic conditions (1% O₂, 5% CO₂, balanced with N₂), for 72 h. Afterwards, PASMCs were fixed with acetone-methanol (1:1) for 5 min at room temperature before apoptotic cells were stained according to manufacturer’s protocol. Finally, cell nuclei were visualized by using Hoechst^®33342 (4 µM; ThermoFisher Scientific, Cat# 62249). Hoechst⁺ (total cell number) and TUNEL-positive (TUNEL⁺) PASMCs were counted by using Leica DMI6000 CS fluorescence microscope and Las X software (Leica, Wetzlar, Germany). Apoptosis was estimated by calculating the ratio between TUNEL⁺ and Hoechst⁺ PASMCs and normalized by daily average.

HaCaT cells were cultivated for 48 h in KGM minimal medium (basal KGM medium supplemented only with insulin and hydrocortisone; Lonza). Then, 20,000 cells per well were seeded in six-well culture plates containing SIPS HDFs. The cells were co-cultured in KGM minimal medium for 8 days, then stained with α-K14 (ab7800; Abcam) and DAPI (Fisher Scientific). Fifteen images per condition were taken at ×50 magnification using a DMI6000 CS fluorescence microscope (Leica) and K14-positive cells were counted. SIPS 1201 HDFs were pre-treated for 4 days with 1201 prior to the experiment. During the co-culture with HaCaT cells, 1201 was not added to the medium.