Cell event senescence green

For analysis of mCherry expression and sorting of viable mCherry+/− cells, single cell suspensions were prepared in PBS supplemented with 2% FBS and 1μg/ml DAPI. Levels of senescence in K562 clonal cell lines were measured using the CellEvent Senescence Green flow cytometry assay kit according to the instructions of the manufacturer (Thermo Fisher Scientific) except that cells were stained with fixable viability dye eFluor 450 (Thermo Fisher Scientific) for 30 min at 4°C prior to fixation according to the manufacturer’s instructions. To analyze β-galactosidase levels in cultures that were a mixture of mCherry+ and wild-type (mCherry−) cells, viable (DAPI-) mCherry+ and mCherry− cells were sort purified prior to fixation in 2% paraformaldehyde, washed extensively with phenol red-free RPMI containing 2% FBS and stored overnight at 4°C prior to staining with CellEvent Senescence Green (Thermo Fisher Scientific). Flow cytometric analysis and sorting was performed at the Flow Cytometry shared Resource Facility at the Icahn School of Medicine at Mount Sinai. Data for analytical purposes were acquired using an LSRFortessa X-20 or FACSCantoII (BD Biosciences). Sorted cell populations were obtained using an Arial II (BD Biosciences) high-speed sorter. All data were analyzed using FlowJo v10.6.1 (Treestar) software.