Senescence β galactosidase sa β gal staining kit

Cell proliferation was assessed using a BrdU cell proliferation kit (6813, Cell Signaling Technology), apoptosis was assessed using the Cell Death Detection ELISAPlus kit (11774425001, Roche), and senescence was assessed using a senescence-β Galactosidase (SA-β gal) staining kit (9860, Cell Signaling Technology) according to the manufacturer’s instructions. Briefly, BC cells were plated at 3000 cell/well in 96 multi-well plates for BrdU and death assays, and 0.125 × 10⁵ cells/well in 24 multi-well plates for senescence assay. Cells were then incubated at 37 °C for 24 h before treatment with the vehicle or drug(s) (alone or in combinations). OD for BrdU incorporation of the S-phase and the level of the nucleosome detected by the immunoreaction were measured at 450 nm and 405/490 nm, respectively, using Paradigm reader. For senescence, cells were photographed on an Olympus IX73 microscope and quantified by counting 200 cells in three different fields for each replicate.

The senescence β-galactosidase (SA-β-gal) staining kit (Cell Signalling) was used to determine cellular senescence following treatments. Cells grown in a 12-well plate format were prepared and fixed for 15 min in accordance with the manufacturer’s instructions before being incubated overnight at 37 °C in freshly prepared SA-β-gal detection solution. SA-β-gal activity was assessed by counting the number of positively stained cells using bright-field microscopy (Leica).

Senescence in cell culture was determined by Senescence β-Galactosidase (SA-β-Gal) Staining Kit (#9860; Cell Signaling) according to the manufacturer’s instruction as previously described (Tang et al, 2013 (link)). The developed blue color on cells was captured with the Zeiss Axiocam HRc camera of Zeiss Observer at the imaging core facility at the Albert Einstein College of Medicine.