Senescence Detection Kit - Biovision

Protocol tips

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	Fix the cells with 0.5 ml of Fixative Solution for 10 - 15 min at room temperature. Add 0.5 ml of the Staining Solution Mix to each well. Cover the plate. Incubate overnight at 37°C.

Protocol tips
Fix the cells with 0.5 ml of Fixative Solution for 10 - 15 min at room temperature. Add 0.5 ml of the Staining Solution Mix to each well. Cover the plate. Incubate overnight at 37°C.

Publication protocol

Cultures of MEFs were obtained at 13.5 d postcoitum. Wild-type, KO, and KO MEFs were incubated ≥24 h before the experiments in medium containing 10% TH-depleted bovine fetal serum by treatment with resin AG-1-X8 (Bio-Rad Laboratories) or in serum-free medium containing Serum replacement-1 (Sigma-Aldrich) and 10 ng/ml human FGF basic. MEFs were normally grown with 20% O, except for the experiment shown in , which was performed under normoxic conditions (3% O). Cells were treated with T3 or with GC-1 (provided by T.S. Scanlan, Oregon Health and Science University, Portland, OR), as indicated in the corresponding figures. Immortalized MEFs from double-KO mice provided by J. Samarut (Ecole Normale Supérieure de Lyon, Lyon, France), TP53KO mice (M. Serrano, Centro Nacional de Investigaciones Oncológicas, Madrid, Spain), and ATMKO mice (E. Callen, National Institutes of Health, Bethesda, MD) were also used. Primary hepatocytes were a gift from A.M. Valverde (Instituto de Investigaciones Biomédicas, Madrid, Spain). pLPCX-THRB, pLPCX-THRA, the C102G, AHT and E457Q THRB mutants (), or the empty vector was used to infect MEFs by retroviral transduction. MEFs were also transduced with pWLZ-Ha-Ras or pWLZ and selected with hygromycin for 5 d (). Transfections with control siRNA (D-001210-01-05; Thermo Fisher Scientific) and NRF1 siRNA (sc-43576; Santa Cruz Biotechnology, Inc.) were performed with Lipofectamine 2000 reagent (Invitrogen). Complementation of TP53KO and ATMKO MEFs was performed by transfection of pcDNA-TP53 or Flag-ATM vectors by the TransIT-TKO transfection method (Mirus Bio LLC). Growth curves were generated following the 3T3 protocol (). Accumulated population doubling levels (PDLs) were calculated as PDL = log(N/N) × 3.33, in which N is the number of inoculated cells, and N is the final number of cells obtained after 3 d. SA-βgal activity was determined as reported by or with the Senescence Detection Kit (BioVision, Inc.). Micrographs were taken in a microscope (TS100F; Nikon) with a digital camera (DS-L1; Nikon), and the percentage of β-galactosidase MEFs was calculated after counting >200 cells.

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