Senescence cells histochemical kit

SAβ-gal activity was measured using a Senescence Cells Histochemical Kit (CS0030, Sigma) according to the protocol supplied by the manufacturer. Briefly, cells were incubated for 24 h with or without addition of 200, 400, and 800 μM H₂O₂ (H1009-100ML, Sigma) or 5 mM N-acetyl-cysteine (NAC) (A9165-25G, Sigma). After fixation with Fixation buffer (2% formaldehyde and 0.2% glutaraldehyde) for 6 min, they were incubated with Staining Mixture at 37 °C overnight. Cells were examined under a ZEISS Primovert microscope (ZEISS, Germany) with ZEISS Axiocam ERc 5s camera (ZEISS, Germany). The cytosol in senescent cells was stained blue. To determine the SA-β-gal activity in uteruses, uteruses were collected from mice, fixed with 10% formaldehyde (v/v) (F1635, Sigma) for 10 min at room temperature, and incubated with Staining mixture for 24 h at 37 °C.

Senescence of cells treated with CFI-400945 and 0.1% DMSO (control) was detected using the Senescence Cells Histochemical Kit (Sigma, USA), which is based on a histochemical stain for beta-galactosidase activity at pH 6. For each cell line, cells were seeded in 6-well plates in triplicates with 100,000 cells per well. Treatment medium was added the next day and incubated for 48 or 72 hrs. After incubation, cells were washed twice with 1X PBS and 1mL of the Fixation Buffer from the kit was added to each well of the plate and incubated at RT for 5 min. Cells were washed with 1X PBS and 1mL of staining solution from the kit was added to each well and incubated at 37°C without CO₂ overnight. The next day, the cells were washed with 1X PBS and the numbers of blue stained cells (which indicate senescent cells) and unstained cells were counted in image J (www.imagej.nih.gov). The percentage of senescent cells was calculated.

For β-galactosidase staining, BM-MSCs of passages P5 to P7 were seeded at a concentration of 2 × 10³ cells/well in 48-well plates and treated as described in the Results section (Section 3.7). After treatment, cells were washed with PBS, fixed, and stained using the Senescence Cells Histochemical Kit according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO, USA). The cells stained for β-galactosidase activity were counted under a light microscope (Olympus, Tokyo, Japan), and the percentage of stained cells was determined for several separated visual fields.