*2.4. Heparanase Treatment of Chondrocytes and Live Cell Imaging*

At P2, chondrocytes were seeded into 12-well plates at or in chamber slides (with 8 chambers) 5200 cells/cm<sup>2</sup> , and treated with complete media supplemented with or without 200 ng/mL of recombinant active human heparanase (Bio-Techne, Abingdon, UK; 20 ng of enzyme results in >50% of optical density (OD) reduction as measured by heparan sulphate release from human syndecan-4) for 48 h. The 12-well plate was placed in a Cell-IQ (ChipMan Technologies, Tampere, Finland) live imaging platform to acquire phase contrast images of all wells, every ten minutes, during the 48-h culture. A built-in analysis software in the Cell-IQ was used to determine the number of cells in each image to produce growth curves of cells treated with heparanase, in comparison to control cells with no enzyme. The mean and standard deviation of the cell counts from three fields of view from three separate repeat wells were taken. After 48 h, the cells were harvested and prepared for multichromatic flow cytometry and real-time quantitative polymerase chain reaction (RT-qPCR) analysis.

The cells within the chamber slides were washed three times with PBS, fixed with paraformaldehyde for 10 min and chamber slides were stored at 4 ◦C until used for immunocytochemistry.
