Live cell observer deconvolution system

The chorioallantoic membrane (CAM) assay was used to assess in vivo angiogenic potential of non-sorted cells and sorted CD34⁺ and CD146⁺ cells. Fertilized eggs (NovoGen, Le Foeil, France) were cleaned with ethanol (70%) and incubated at 37 °C in 60% humidity. Three days later, 2 ml of albumen was aspirated from the egg with a syringe and a window was created which was covered again and returned to incubation. On day 8 of incubation, polypropylene rings were placed on the CAM and cells (75,000 cells) or PBS were placed in the center of the ring, as described previously [28 (link), 29 ]. The eggs were incubated for a further 2 days, when images from each ring were taken using the Zeiss AXIO ZoomV16 microscope at 40×. Binary images were generated by Image J and angiogenesis was quantified using AngioSys2.0 software (Cellworks, Buckingham, UK).
For in vitro angiogenesis experiments, CD146⁺ and endothelial cells were stained with PKH26 (20 μM) and PKH67 (20 μM) (both from Sigma-Aldrich), producing red and green fluorescence signals, respectively. Once labeled, cells were resuspended in EGM-2 medium and seeded on ibiTreat μ-Slides (IB-81506; Thistle Scientific) coated with matrigel. Pictures were taken using a Zeiss Live Cell Observer/Deconvolution system.

For time-lapse imaging, E6.5 explants prepared from CAG-GFP chicken embryos were mounted onto nitrocellulose filters and cultured skin side down in a 6-well dish containing 2 ml standard or supplemented medium at 37°C, 5% CO₂. Real-time imaging was performed using the Zeiss Live Cell Observer/Deconvolution system, imaged every 10 minutes for the duration of the experiment, and analysed using ZenBlue2012 (Zeiss) software.
For the PIV analyses [77 ,78 (link)], microscope image frames of skin showing fluorescent (red and green) cells were superposed in one image to achieve higher density. PIV was conducted using MatPIV, implemented in Matlab, with global, local, and signal-to-noise filter. Interrogation areas (32 × 32 pixel size) with 50% overlap were chosen. PIV yields displacement (velocity) fields for every pair of consecutive frames, which were used to derive velocity and angle kymographs as well as streamlines.