38HK cells were transduced with p19INK4d and cultured for 72 h. The NIH 3T3 feeder layer was removed with PBS/EDTA from 38HK cultures prior to senescence analyses.
Senescence was assessed using the Senescence β-Galactosidase Staining Kit at pH 6 following the manufacturer’s instructions (Cell Signaling Technology). For SAHF staining, 38HK cells were layered on slides coated with polylysine and fixed in 4% paraformaldehyde in PBS (pH 7.4) for 15 min at room temperature, and permeabilized with 0.1% Triton X-100 in PBS for 15 min (60 (link)). Cells were incubated with H3K9me3 antibody (abcam; ab1220) for 2 h at room temperature, followed by incubation with Alexa Fluor 555-conjugated secondary antibody for 1 h at room temperature, and mounted using Vectashield Antifade Mounting Medium with DAPI. The slides were visualized using a Nikon Eclipse Ti wide-field inverted fluorescence video microscope. The images captured were analyzed by NIS-Element software from Nikon.
Senescence was assessed using the Senescence β-Galactosidase Staining Kit at pH 6 following the manufacturer’s instructions (Cell Signaling Technology). For SAHF staining, 38HK cells were layered on slides coated with polylysine and fixed in 4% paraformaldehyde in PBS (pH 7.4) for 15 min at room temperature, and permeabilized with 0.1% Triton X-100 in PBS for 15 min (60 (link)). Cells were incubated with H3K9me3 antibody (abcam; ab1220) for 2 h at room temperature, followed by incubation with Alexa Fluor 555-conjugated secondary antibody for 1 h at room temperature, and mounted using Vectashield Antifade Mounting Medium with DAPI. The slides were visualized using a Nikon Eclipse Ti wide-field inverted fluorescence video microscope. The images captured were analyzed by NIS-Element software from Nikon.
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