Fitc ki 67

Confluent HUVECs were either treated or not treated with native LDL or Mox-LDL at a final concentration of 100 μg/mL for 24 hours. Cell death was detected using the LIVE/DEAD Fixable Red Dead Cell Stain Kit (Molecular Probes; Cat. number L-23102) following manufacturer's instructions. HUVECs were harvested and stained with the near-IR Dead Cell Stain which is excited at a 633/635 nm wavelengths. For proliferation analysis, BD Pharmingen Ki67 kit was used and the assay was also performed according to manufacturer's instructions. In brief, after staining with the Red Dead Cell Stain, cells were fixed, permeabilized, and then stained with FITC-Ki67 (BD Pharmingen, San Diego, CA, USA; Cat. number 612472) for 20 min at 4°C. Cells were then analyzed using BD FACSCanto II flow cytometry, and data were analyzed using FlowJO (Tree Star, inc.©). The experiments were performed in duplicate and the percentages of proliferating and dead cells were averaged between the duplicates.

Sorted HSCs were fixed and permeabilized using Cytofix/Cytoperm plus kit (BD), according to the manufacturer's instruction. Fixed cells were then stained overnight with FITC Ki67 (BD) at 4 °C, and 10 min by Hoechst 33342 (Invitrogen).