Islets were dispersed using 1 ml of Accumax (#07921, STEMCELL Technologies) containing deoxyribonuclease I (2 U/ml) and Pluronic F-127 for 15 min at 37°C. Single cells were stained with 25 μM Newport Green PDX for 90 min at 37°C. Cells were washed, resuspended in 1 ml of phosphate-buffered saline, and incubated at 37°C for 30 min. Cells were fixed with 2% paraformaldehyde for 10 min and subsequently permeabilized with Transcription Factor Staining Buffer Set (Invitrogen). Intracellular staining was carried out in perm/wash buffer containing Ki67-PE (phycoerythrin) (clone 16A8, BioLegend) and histone H3 (pS28) Alexa Fluor 647 (clone HTA28, BD Biosciences) overnight 4°C. After staining, the cells were washed, filtered, and acquired on a FACSCanto II cytometer (BD Biosciences). Data were analyzed using FlowJo software (Tree Star).

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