C9100 02 emccd camera

Tube-formation assays were performed using modifications to standard procedures described previously [50 (link)]. Matrigel (Corning) was thawed overnight, on ice at 4°C and 50 μl transferred to the desired wells of a 96 well, clear bottom plate. The Matrigel was then allowed to solidify at 37°C for at least 30 minutes before use. A flask of MECs cultured to 90-100% confluency in EGM-2MV media were then trypsinized, separated into two tubes, and pelleted by gentle centrifugation. The media was aspirated from each tube and the cells from one tube were resuspended in HepG2 TCM and the other in serum free DMEM as a negative control. Cells were mixed with peptide or DMSO solutions as indicated (DMSO was used for the negative control) and 15,000 cells plated in each of the Matrigel coated wells. The plate was then incubated at 37°C in a cell culture incubator for 18 hours after which the cells were fixed in 10% neutral buffered formalin. Images were taken using an Olympus IX81 with a 2x objective and a Hamamatsu Photonics C9100-02 EMCCD camera. As treatment at all concentrations of peptide completely disrupted tube formation, quantification of tube networks was unnecessary.

Three dimensional z-stacks with a z-resolution of 150 nm were taken at Nikon Imaging Center at Bioquant Heidelberg, using a Perkin Palmer ERS6 spinning disk microscope. All stacks were measured with a Nikon Plan Apo λ 100x/NA 1.45 oil immersion objective and a Hamamatsu C9100-02 EMCCD camera (1000 x 1000 pixel, 8 μm pixel size). Lasers of 405 nm, 488 nm, 561 nm and 640 nm excitation wavelength were used for fluorochrome activation such as DAPI DNA counterstain and Alexa stained antibodies. For green (527/55), blue/red (455/60 and 615/70) and far red (485/60 and 705/90) dual pass filters were available. The number of pictures taken per stack was picked manually in order to measure individually over the whole cell nucleus. After microscopy image stacks were de-convolved with Huygens Remote Manager software, to improve picture quality.