Dapi prolong

To assess senescence in vitro, cellular SA-β-Gal activity was measured as described previously⁹⁴ using the Cell Signaling Technology Senescence β-Galactosidase Staining Kit. Briefly, CD24+ or – cells were seeded on 8-well chamber slides at 1 × 10⁴ cells/cm² and allowed to grow for 14 days. Cells were then washed in PBS (pH 7.4) and fixed with 1X fixative solution for 5 min, then washed three times using PBS. Cells were then incubated in 1X SA-β-Gal staining solution at 37 °C for 16 hr. Cells were washed in ice-cold PBS and mounted with DAPI ProLong (ThermoFisher) staining nuclei for cell counting. In blinded fashion, ten images per well were taken from random fields using fluorescence microscopy (Nikon Eclipse Ti) and SA-β-Gal-positive cells were counted and reported as a percentage of total cells.

Cells were fixed 10 min in 4% paraformaldehyde, blocked 30 min in PBS with 1% BSA and labelled overnight with M75 antibody in blocking solution⁴⁷ (link). After washing with PBS +0.02% Tween20, cells were labelled with anti-mouse conjugated with AlexaFluor-488 and then with DAPI + ProLong (P36931, ThermoFisher Scientific).
Images were taken by a FV1000 confocal microscope (Olympus, Tokyo, Japan), using a (Olympus) planapo objective 60x oil A.N. 1.42. Excitation light was obtained by a Laser Dapi 408 nm for DAPI, an Argon Ion Laser (488 nm) for Alexa 488, DAPI emission was recorded from 415 to 485 nm, AlexaFluor-488 emission was recorded from 525 to 550 nm.