Senescence β galactosidase kit

Primary old cardiac fibroblasts were seeded on 35 mm culture dishes. After cells were grown to 70% confluence, serum-free media or CM was added for 48 hours, followed by β-gal staining using Senescence β-galactosidase kit (#9860, Cell Signaling) per the manufacturer’s instructions. Images were captured using Nikon microscope. The percentages of SA-β-gal⁺ cells were calculated.

The ratio of β-galactosidase in the BJ1 cells was measured using the senescence β-galactosidase kit (cell signaling) according to the manufacturer's protocols. BJ1 fibroblasts overexpressing miR-200c (F200c) or control were cultured in 10% nuserum DMEM media for four days.

Telomere length was quantitated using TeloTAGGG Telomere Length Assay (Roche, Basel, Switzerland) according to the written protocol. β-galactosidase staining was performed using the Senescence β-galactosidase kit (Cell Signaling, Danvers, MA, USA).

Cells were seeded in 6‐well plates before treatment with volasertib (0–20 nm) for 24 h. Seventy‐two hours later, cells were fixed and stained at pH 6.0 using a senescence β‐galactosidase kit (Cell Signaling, no. 9860). Plates were incubated with X‐gal staining solution overnight at 37 °C in a dry incubator without CO₂. Using a transmitted‐light microscope (Olympus BX41), equipped with a Leica DFC450C camera, blue staining was visualized in three random nonoverlapping fields using a 10x objective with a 10× eyepiece for quantification. The percentage of senescent cells (morphological changes combined with blue staining) was determined using imagej software.

Cellular senescence was evaluated by β-galactosidase activity using β-galactosidase Senescence Kit (#9860, Cell Signaling Technology). Briefly, cells were fixed for 15 min followed by washing in PBS twice. Then fixed cells were incubated with β-galactosidase staining solution at 37 °C in a dry incubator (no CO₂) at least overnight. The cells were then observed under a microscope for the development of blue color.