Phalloidin ritc

Tumor and matched normal Lin-EpCAM-CD73+CD90+ cells were harvested and seeded in glass slide chambers coated with 0.1% gelatin at 50,000 cells/well. 48 hours after seeding, cells were treated with 50 ng/ml recombinant human Jagged-1 (Peprotech, Rocky Hill, NJ, USA) and/or 10 ng/ml recombinant human TGF-β1 (Invitrogen) for three consecutive days. Media was saved and stored at −80 °C. Following treatment, cells were fixed with 4% paraformaldehyde (Sigma Aldrich) for 15 minutes. The permeabilization of cells was carried out with 0.1% Triton X-100 (Sigma Aldrich), followed by lock for 1 hour in 2% BSA (Sigma Aldrich) and stained with αSMA-FITC (Sigma Aldrich), Phalloidin-RITC (Molecular Probes, Eugene, OR, USA) and Hoechst 33342 (Invitrogen) for 2 hours. Samples were imaged using a Zeiss laser scanning microscope 710.

On day seven, EGM2 in the microfluidic chips was replaced with 200 μl PBS (Invitrogen) prior to fixation with 4% paraformaldehyde for 15 minutes. After three washings with PBS, the cells were permeabilized with 0.1% Triton X-100 (Sigma Aldrich) and blocked for one hour in 2% BSA (Sigma Aldrich) in PBS. Subsequently, the cells were either directly labeled with αSMA-FITC Clone 1A4 (Sigma Aldrich), Phalloidin-RITC (Molecular Probes, Eugene, Oregon, USA) and Hoechst 33342 (Invitrogen) in PBS, or incubated overnight with goat PECAM-1 (Santa Cruz Biotechnologies, San Diego, CA, USA), and after washing incubated with donkey ant-goat Alexa 546 secondary antibodies 1:500 (Molecular Probes) and directly labeled antibodies. The microfluidic chips were imaged on a Zeiss confocal laser scanning microscope 710. For signal quantification, the mean intensity on an area of 1.5 mm * 1.5 mm was measured per well.