Mouse islets were handpicked with a stereomicroscope after collagenase digestion of pancreases (Rui et al., 2016 (link)). Single-cell suspensions were prepared after a second collagenase digestion. Cells were stained with FluoZin-3 (Invitrogen) and TMRE (Life Technologies) and anti-CD45 mAb (eBioscience). In some experiments, single islet cells were stained with anti-insulin (R&D Systems) and anti-glucagon (Abcam) antibodies labeled with fluorescein isothiocyanate (FITC), PD-L1 (BioLegend), or CD40 (BioLegend) and analyzed with LSRFortessa (BD). For purification, β cells stained with TMRE and FluoZin-3 were sorted using an FACSAria II (BD).
In other studies, β cell subpopulations were sorted from 8- to 10-week-old NOD mice (n = 6–8 per experiment), and aldehyde dehydrogenase (ALDH) activity was detected by flow cytometry after processing the cells using an ALDEFLUOR kit (STEMCELL Technologies) according to the manufacturer’s instructions.
In other studies, β cell subpopulations were sorted from 8- to 10-week-old NOD mice (n = 6–8 per experiment), and aldehyde dehydrogenase (ALDH) activity was detected by flow cytometry after processing the cells using an ALDEFLUOR kit (STEMCELL Technologies) according to the manufacturer’s instructions.