Senescence β galactosidase activity assay kit

β-Galactosidase activity in senescence was measured using the senescence β-galactosidase activity assay kit (Cell Signaling Technology, Beverly, MA, USA) according to the manufacturer’s instructions. Cells were washed with cold 1× PBS. After removal of the wash solution, cold 1× Senescence Cell Lysis Buffer with 1.0 mM PMSF was added. After incubation on ice for 5 min, the cell lysate was centrifuged at 14,000 rpm for 5 min at 4 °C. The supernatant was transferred to a fresh tube, and the total protein concentration of each cell lysate was determined using a protein assay. Fifty microliters of the cell lysate was transferred to a 96-well plate, and 50 μL of freshly prepared 2× assay buffer (10 mM BME in 2× senescence reaction buffer) was added. The plate was incubated at 37 °C in the dark for 2 h. Fifty microliters of the reaction mixture was transferred to a 96-well black opaque plate, and 200 μL of senescence stop solution was added to each well. The 96-well plate was read with a fluorescence plate reader set with excitation at 360 nm and emission at 465 nm.

The Senescence β-Galactosidase Activity Assay Kit (fluorescence, plate-based; #23833; Cell Signaling Technology, Danvers, MA, USA) was used for SAβgal staining according to the manufacturer’s protocol. Cells were stained overnight at 37° C in a room CO₂ incubator air. The cells were washed with phosphate-buffered saline (PBS) (−) and stained with Hoechst 33342 (H342; Dojindo, Kumamoto, Japan). Imaging and quantification of stains were performed using a BZ-X700 microscope (Keyence, Osaka, Japan).

Human B-ALL cell lines (10⁶ cells/well in a 12-well plate) were cultured in unconditioned, bone marrow stromal cell-, or adipocyte-conditioned media (depending on the experiment) for 24 h. After the incubation period, cells were harvested and lysates prepared. β-Galactosidase activity was determined using the Senescence β-Galactosidase Activity Assay Kit from Cell Signaling Technology (cat# 23833 S) per the manufacturer’s protocol. Relative Fluorescence Units (RFU) of 4-MU were measured at an excitation wavelength of 360 nm and an emission wavelength of 465 nm using the Synergy Neo2 Hybrid Multi-Mode Plate Reader (BioTek, Winooski, VT).