Columbus image data storage and analysis system

Cells were plated on gelatin-coated 96 well plates at a density of 30,000 cells cm⁻² and stained with anti-proliferating cell nuclear antigen (PCNA) antibody (abcam, ab29, 1:200) when the cells are at 60–70% confluent. As for CAVSMC and dVSMC, cells were cultured with or without Dox before the assay. To detect cell nucleus and cytoplasm, cells were simultaneously stained with Hoechst and Whole Cell Stain Blue. For counting PCNA positive cells, 49 fields per well were imaged at 20x magnification using Cellomics ArrayScan VTI. Subsequent image quantification was performed automatically using the Columbus Image Data Storage and Analysis System or HCS Studio Cell Analysis Software (Thermo Scientific).

Cells were plated on gelatin-coated 96 well plates at a density of 30,000 cells cm⁻². In situ cell death detection was performed three days after passage by TUNEL technology as described by the manufacturer (In situ Cell Death Detection Kit or In situ Cell Death Detection Kit, TMR red, Roche). As for human VSMCs, cells were cultured with or without Dox before the assay and apoptotic cells were also detected by staining with anti-Cleaved Caspase-3 antibody (Cell Signaling Technology, #9664, 1:400). To detect cell nucleus and cytoplasm, cells were simultaneously stained with Hoechst and Whole Cell Stain Blue. For counting apoptotic cells, 49 fields per well were imaged at 20x magnification using Cellomics ArrayScan VTI. Subsequent image quantification was performed automatically using the Columbus Image Data Storage and Analysis System or HCS Studio Cell Analysis Software (Thermo Scientific).