Senescence Cells Histochemical Staining Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
The staining of senescent cells is pH dependent. Therefore, the cells cannot be incubated in a CO2 enriched atmosphere during the staining step	Stain cells with staining mixture (containing X-gal) at 37°C for 24 h

Upstream tips
The staining of senescent cells is pH dependent. Therefore, the cells cannot be incubated in a CO2 enriched atmosphere during the staining step
Protocol tips
Stain cells with staining mixture (containing X-gal) at 37°C for 24 h

Publication protocol

Senescence was determined using the Senescence Cells Histochemical Staining Kit (Sigma-Aldrich, CS0030). Briefly, cells were incubated with 1x Fixation Buffer for 7 min at RT. Next, cells were washed and incubated with staining mixture (containing X-gal) at 37°C for 24 h. Subsequently, cells were counterstained with Nuclear Fast Red (BDH, 34209). The percentage of senescence-associated GLB1-positive cells was quantified using ImageJ software. Cell cycle analysis was performed by flow cytometry using BD FACScan (BD, Franklin Lakes, NJ, USA) as previously described.68 Results were analyzed with FCS Express 4 Flow software. To detect DNA damage, a comet assay was performed as previously described.69

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