Figure 1

Effect of papain on cell viability (panel A) and lactate dehydrogenase release (panel B). To test cell viability, HUVEC were seeded into fibronectin coated microtiterplates and cultured in EGM. After 24 hours medium was changed to EBM and cells were cultured for 10 hours in EBM containing 10 ng/mL VEGF or 10 ng/mL VEGF and 10 μg/mL papain. Cells were stained using life cell staining calcein-AM. Cells were photographed at 4 fold magnification using a Nikon Eclipse Ti, the FITC filter set of the instrument and a Nikon Digital Sight DS-Fi1C CCD camera. Integrity of the monolayer and viability of cells was assessed visually. To investigate lactate dehydrogenase release, HUVEC were seeded into fibronectin coated 96-well microtiterplates and cultured in EGM. After 24 hours medium was changed to EBM containing 10 ng/mL VEGF or 10 ng/mL VEGF and papain at concentrations as indicated. After 48 hours, released lactate dehydrogenase was determined using a 96-Cytotoxassay kit. Data are shown as mean ± standard error of measurement. Monolayers of papain treated cells are intact and cells are viable as demonstrated by calcein uptake, metabolization and retention (panel A). No increase of LDH in the supernatant was apparent at enzyme concentrations of up to 25 μg/mL (panel B).