Evos fi fluorescent microscope

To analyse the morphological changes on F-actin organization in hPSCs, cells were seeded into 24 well plate at a seeding density of 4,000 cells/well and starved for 24 h. hPSCs were then incubated 5 ng/ml TGF-β for 24 h and then they were washed three times with DPBS (Lonza) and fixed in 4% formaldehyde (Sigma Aldrich) in DPBS for 20 minutes. After permeabilizing the cells with 0.1 M Triton X-100 (Sigma Aldrich) for 5 minutes, F-actin was stained with phalloidin (Life Technologies, Carlsbad, USA) at concentration of 250 ng/ml for 60 minutes. Next, cells were washed with DPBS and then mounted with DAPI (Sigma Aldrich) containing mounting medium. Images were captured using an EVOS FI fluorescent microscope (Life Technologies) at excitation/emission 385nm/460nm (DAPI) and excitation/emission 540nm/565nm (F-actin).

hPSCs were seeded into 24 well plates at a seeding density of 10,000 cells/well. After overnight, cells were starved for 24 h and treated with 5 ng/ml TGF-β and FGF2 at 250 ng/ml (low) or 500 ng/ml (high). After 48 h incubation, cells were fixed with acetone:methanol (1:1) for 30 minutes at -20 °C followed by drying at RT and rehydration with PBS. To analyse protein expression of α-SMA and collagen-1, cells were incubated with respective primary antibody for 1 h at room temperature followed by fluorescence labeled secondary antibody for 30 minutes in dark condition. Finally, cells were mounted with DAPI-containing fluoroshield (Sigma Aldrich). Images were made using an EVOS FI fluorescent microscope (Life Technologies) at excitation/emission 385nm/460nm (DAPI), excitation/emission 485nm/530nm (GFP) and excitation/emission 590nm/617nm (RFP). Images were analysed using ImageJ software (NIH).