Vybrant dio cell labelling solution

To evaluate angiogenesis (tube formation) in vitro, MPECs were seeded onto Matrigel; total tube length was assessed after 6 h. To investigate in vivo angiogenesis, Matrigel supplemented with either VEGF or basic fibroblast growth factor (bFGF) was mixed with MPECs loaded with Vybrant® DiO cell labelling solution (Life Technologies) and injected into C57BL/6 mice anaesthetized using isoflurane. Following sacrifice, plugs were excised, snap-frozen, and cryosectioned for immunostaining with an anti-CD31 antibody and imaged by confocal microscopy. Capillary formation was assessed by determining the percentage of CD31-positive Vybrant-labelled MPECs compared with the total number of CD31-positive cells within four randomly selected fields of view per plug. Animals were studied in accordance with Directive 2010/63/EU of the European Parliament.

HaCaT cells were stained with Vybrant DiO cell-labelling solution (Life Technologies, Waltham, MA, USA) at 1:200, as per manufacturer’s instructions. Stained HaCaTs were then seeded at 160,000 cells/mL together with 220,000 cells/mL SCC-12 into a 24-well plate 24 h prior to start of experiment.