Senescence Cells Histochemical Staining Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 2 × 10^4 cells and pretreat with agents.	After72 h of pretreatment , fix cells with 2% paraformaldehyde/glutaraldehyde. Add 1 ml of the Staining Mixture per well and incubate at 37 °C without CO2 until the cells are stained blue

Upstream tips
Seed 2 × 10^4 cells and pretreat with agents.
Protocol tips
After72 h of pretreatment , fix cells with 2% paraformaldehyde/glutaraldehyde. Add 1 ml of the Staining Mixture per well and incubate at 37 °C without CO2 until the cells are stained blue

Publication protocol

HepG2, Hep3B, A-498, HCT-116, HCT-116/p53−/−, and A549 cells were plated in 6-well plates at a density of 2 × 104 cells per well and treated with the respective agents. After 72 hours (HepG2 and Hep3B) or 96 hours (A-498, HCT-116, and A549), cells were washed with PBS and fixed in 2% paraformaldehyde/glutaraldehyde before the staining with X-gal solution according to the manufacturer's instructions (Senescence Cell Histochemical Staining Kit, Sigma-Aldrich). After 24 hours of incubation at 37°C, nuclei were stained with 2 μg/mL Hoechst 33342 (Invitrogen). The percentage of β-galactosidase–positive cells was determined by counting nuclei and cell bodies with a blue precipitate (Olympus IX50, analySIS, Soft Imaging System). For each time point and concentration at least 100 cells were counted.

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