Igg2bκ isotype control

The cell surface expression of ITGα5β1 and αvβ5 was determined by flow cytometry. 1 × 10⁶ cells were harvested using TrypLE. After washing, JJ012 cells were stained with 5 µL of Alexa Fluor 488-labeled anti-ITGα5β1 antibody (volociximab, Novus Biologicals, Centennial, CO, USA) or IgG isotype control (Novus Biologicals, Centennial, CO, USA) in 45 µL Flow Cytometry Staining Buffer (ThermoFisher Scientific, Waltham, MA, USA) for 30 min on ice in the dark. HT1080 cells were stained with 5 µL of BV421-labeled anti-ITGαvβ5 antibody (BD Biosciences, San Jose, CA, USA) or IgG2bκ isotype control (BD Biosciences, San Jose, CA, USA) under the same conditions. After a single wash with staining buffer, the cells were fixed in 4% paraformaldehyde (ThermoFisher Scientific, Waltham, MA, USA) and read on an LSR-II analyzer (BD Biosciences, San Jose, CA, USA).

To analyze surface markers on freshly isolated hAC, cartilage tissue was directly digested by means of collagenase overnight, without pronase. In case of cultured hAC, cells were detached with PBS-buffered EDTA (5 mM). hAC were stained with anti-CSV (clone 84-1, abnova, #H00007431-MF08) or IgG2bκ isotype control (BD Pharmingen) for 40 min. A minimum of 2×10⁴ cells was analyzed on a Becton Dickinson FACSCalibur flow cytometer (BD Biosciences) with dual-laser technology using the corresponding software CellQuest version 5.2.1. A maximum of 1% isotype control-positive cells was accepted. Cancer cell lines HeLa (low CSV levels; mean: 11.8%±4.2) and SaOs-II (mediate/high CSV levels; mean: 34.4%±8.8) were used to validate the anti-CSV antibody and the staining protocol (Supplementary file 1). While CVS was found to be degraded by trypsin and accutase treatment, no influence was observed in case of collagenase relative to EDTA (Supplementary file 1).