Senescence cells histochemical staining kit

Sirt6^flox/flox (Sirt6^f/f, Sirt6^tm1.1Cxd/J) and Tie2-Cre (Tie2^cre/+, B6.Cg-Tg(Tek-cre)1Ywa/J) mice were purchased from the Jackson Laboratory (Sacramento, CA, USA). Sirt6^f/f mice were backcrossed with C57BL/6 mice to produce congenic strains. Then, Sirt6^f/f mice were crossed with Tie2-Cre mice expressing Cre recombinase under the control of a Tie2 promotor to generate mice (Sirt6^f/f/Tie2^cre/+) deficient of endothelial cell SIRT6. Mice were genotyped by PCR according to the provider’s instruction. Sirt6^f/f mice from the same litters were used as controls against Sirt6^f/f/Tie2^cre/+ mice. C57BL/6 mice were obtained from Orient Bio Company (Seongnam, Korea). To induce senescence of aorta, male mice (8–9 weeks old) were intraperitoneally injected with 25 mg/kg PQ (Sigma-Aldrich, St. Louis, MO, USA). After 3 d, mice were anesthetized and sacrificed. After systemic perfusion with PBS, the thoracic aorta was excised and fixed with formalin or fixation buffer from the Senescence Cells Histochemical Staining Kit (Sigma-Aldrich). Animal care and experimental procedures were performed following approval from the Institutional Animal Care and Use Committee of CHA University (Approval No. IACUC160065).

Isolated CAFs (at passage 3) were seeded in DMEM supplemented with 10% FBS at a density of 20.000 cells per well in six-well plates and left for attachment and spreading for 24 h before irradiation. Five days post-irradiation, cultures were washed and fixed for 5–7 min at 20°C with paraformaldehyde (2%) and stained for β-galactosidase (5-bromo-4chloro-3-indolyl-B-D-galactopyranoside). Staining was achieved following instructions from the manufacturer; “Senescence Cells Histochemical Staining Kit” (# CS0030, Sigma-Aldrich). Randomly selected fields were photographed at 1000× magnification, using an Idea SPOT digital camera.

Senescence Cells Histochemical Staining Kit was purchased from sigma (cs0030). Cells were stained according to the manufacturer’s instructions.

Senescent cells were identified using the Senescence Cells Histochemical Staining kit (Sigma). Briefly, EPCs were washed in PBS, fixed for 7 minutes at room temperature, washed again and incubated for 16–18 hours at 37°C (no CO₂) with X-gal chromogenic substrate. The cells were then washed with PBS, added with DMSO for dissolving the stain, and incubated at 37°C for 30 min, followed by measurement of absorbance at 620 nm.

The assay was performed using a Senescence Cells Histochemical Staining Kit (CS0030, Sigma) according to manufacturer’s instructions. Briefly, 72 h after incubation in the presence of 4-OHT, MEFs were fixed for 7 min in fixation buffer, washed in PBS and stained with staining mixture overnight at 37 °C without CO₂. The cells were observed under a bright field microscope (DM IL LED, Leyca). The percentage of β-galactosidase-positive cells was calculated by counting blue-stained cells and the total number of cells.