Fitc labelled phalloidin

To determine protein cellular localization for SATB1 and E-cadherin, immunofluorescence was performed using an A1Si laser-scanning confocal microscope. Briefly, BIU-87 and T24 cells transfected with pcDNA3.1-SATB1 and pGenesil2-SATB1-shRNA expression vectors were seeded onto glass coverslips and placed in 24-well plates. Cells were fixed with 4% paraformaldehyde for 30 min at 37°C after incubation for 3 days, and then washed with PBS twice for 10 minutes each. The fixed cells were permeabilized with 0.3% Triton- X 100 for 15 min, blocked in 10% goat serum for 1h, and then washed with PBS twice for 10 minutes each. Then the cells were incubated with primary antibody as follows: rabbit polyclonal antibody SATB1, and E-cadherin(Abcam Inc., Cambridge, CA, USA) (1:50) at 4°C overnight followed by detection with Cy5-conjugated secondary antibodies and FITC-labelled phalloidin (5μg/ml)(sigma) for 1 hour at room temperature in the dark. DAPI was subsequently used to stain nuclei. Images were collected using a Nikon A1Si laser-scanning confocal microscope (Nikon, Japan).

Cells on coverslips were rinsed 3 times with PBS containing 0.9 mM Ca²⁺ and 0.49 mM Mg²⁺ (Sigma D8662) and fixed with 1% paraformaldehyde in 0.1 M HEPES (pH 7.4) for 15 min at room temperature. After being permeabilised cells were blocked with 1% BSA in PBS at room temperature for 30 min. FITC labelled phalloidin (Sigma) was added and incubated for 1 hr in the dark. Cells were washed, then left to air dry before mounting with vector shield containing DAPI and left to set at 4°C. Samples were examined with a Leica light microscope. Cell area was measured using ImageJ. 10 cells were measured from each isolate (n = 30 per time point).

The morphology of the cells on top of the scaffolds was observed after 1, 3 and 7 days of culture. 16.000 hADSCs in 1 mL of α-MEM culture medium (no glutamine, 5% human serum, 1% P/S) were cultured in direct contact with scaffolds. Culture medium was refreshed at day 2, 4 and 6. The control was 10 mm diameter glass coverslips (Marienfeld, Lauda-Konigshofen, Germany) in a 48-well plate.
At each time point, the cells were fixed with 4% (w/v) para-formaldehyde solution in PBS (AlfaAesar, Haverhill, MA, USA) for 15 min. Next, cells were permeabilized with 0.1% (v/v) Triton X-100 (Sigma Aldrich, St Louis, MO, USA) for 10 min. The nonspecific binding sites were blocked by incubating the scaffolds in Phosphate Buffered Saline (PBS) (Medicago AB, Uppsala, Sweden) containing 3% (wt/v) Bovine Serum Albumin (BSA, Sigma Aldrich, St Louis, MO, USA) for 45 min. Subsequently, the samples were incubated for 45 min with stains diluted in PBS. Cytoskeleton and nucleus were stained using FITC-labelled phalloidin (1:500) (Sigma Aldrich, St Louis, MO, USA P1951) and 4’,6-Diamidino-2-phenylindole dihydrochloride (1:2000) (DAPI, Sigma Aldrich, St Louis, MO, USA D9542), respectively. Finally, the samples were rinsed with PBS–BSA 0.5% and pure water and observed using a LSM800 confocal microscope (Zeiss, Iena, Germany).