Osteopontin pe

The quantification of VSMC osteoblast-like cell transdifferentiation was performed using VSMC derived from the AA&R of male C57Bl/6 mice. In vitro VSMCs were stimulated with either 40ng/mL oxLDL or were co-cultured with LYVE1 macrophages derived from Apoe^−/− Myh11-CreERT2, ROSA26 STOP-flox eYFP^+/+ mice on HCD using Transwell cell culture inserts for 7 days. The quantification of VSMC osteoblast-like cell transdifferentiation was performed via a flow cytometry analysis of RUNX2 DyLight 405 (NOVUS), alkaline phosphatase APC (NOVUS), osteopontin PE (R&D), collagen 8 and Mac-2 PE/Cyanine7 (Biolegend, San Diego, CA, USA), and Brilliant Violet 650™ anti-mouse F4/80 after excluding dead cells via LIVE/DEAD Fixable Near-IR Dead Cell Dye staining (Thermo Fisher). Samples were acquired in CytoFLEX (Beckman Coulter) and were analyzed with FlowJo software (TreeStar, Version 10.5.3, Woodburn, OR, USA).

VSMC derived from AA&R of male C57Bl/6 and Apoe^−/− C57Bl/6 mice were stimulated with either 40 ng/mL oxLDL or/and 150 ng/mL of GDF10 for 7 days. Quantification of VSMC osteoblast-like cell transdifferentiation was performed via flow cytometry analysis of RUNX2 DyLight 405 (NOVUS), Alkaline Phosphatase APC (NOVUS), osteopontin PE (R&D), GDF10 Alexa 700 (Thermo Fisher) and CD68 PerCP/Cyanine5.5 (Biolegend, San Diego, CA, USA), excluding dead cells via LIVE/DEAD Fixable Near-IR Dead Cell Dye (Thermo Fisher, Waltham, MA, USA). Samples were acquired in CytoFLEX (Beckman Coulter, Brea, CA, USA) and analyzed with FlowJo software (TreeStar, Version 10.5.3, Ashland, OR, USA).