Live dead fixable near ir dead cell dye staining

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).

Quantification of VSMC transdifferentiation was performed using VSMC in passage 1. In vitro VSMC were stimulated with either 40 ng/mL oxLDL (Thermo fisher), Z-VAD-FMK 10 μM (InvivoGen) or 100 ng/mL Colchicine (Sigma-Aldrich), or 10 ng/mL of IL-1β for 7 days or co-cultured with monocytes derived from male C57Bl/6 mice using Transwell Cell Culture Inserts for 7 days. VSMCs were co-cultured with monocytes or monocytes activated with oxLDL upon direct supplementation of 40 ng/mL oxLDL (Thermo Fisher) to well cell culture inserts (pore size 0.02 μm) in the trans well plates for 7 days. The direct supplementation of oxLDL with a known diameter size of more than 20 nm [47 (link)] ensured monocytes restricted oxLDL activation since oxLDL was retained in the trans well inserts with a pore size of 0.02 μm. Quantification of VSMC transdifferentiation was performed via flow cytometry analysis of anti-mouse CD68 PerCP/Cy5.5 (Biolegnd), anti-mouse MAC2 PE/Cy7 (Biolegnd), anti-mouse F4/80 Brilliant Violet 650™, α-SMA Alexa Fluor 488, SM22α⁺ Alexa Fluor 700 after excluding dead cells via LIVE/DEAD Fixable Near-IR Dead Cell Dye staining (Thermo fisher). Samples were acquired in Gallios flow cytometer (Beckman Coulter) and analyzed using FlowJo software (TreeStar, Version 10.0.8r1, Ashland, OR, USA).