Senescence galactosidase staining kit

To determine rate of DNA synthesis, cells were pulsed with 10 µM EdU (5-ethynyl-2’-deoxyuridine; Thermo Fisher Scientific, Grand Island, NY) for 1 hour. Labeled cells were trypsinized, washed in PBS and fixed in 4% paraformaldehyde for 20 min at RT and washed in PBS. Fixed cells were further stained by incubating in the staining cocktail (4 mM CuSO4, 100 mM sodium ascorbate and 4.8 µM Alexa Flour 647 Azide triethylammonium (Thermo Fisher Scientific)) for 20 min at RT followed by washes in PBS. Cells were re-suspended in PBS and counterstained with 5 µg/ml of DAPI. Analyses were performed on flow cytometer Fortessa B at Roswell Park Cancer Institute FACS facility, using BD FACS Diva Software (BD Biosciences). Data were collected for 20000 cells and analyzed with FCS Express 4 (De Novo Software, Glendale, CA).
SA-β-gal staining was performed using Senescence-galactosidase staining kit (Cell Signaling Technology, Danver, MA) according to manufacturer’s protocol. Photos were taken under bright field microscope.

SA-β-gal activity also was evaluated with senescence galactosidase staining kit (Cell Signaling Technologies, USA). 2D-MSCs grown on 6 wells plate (TPP) for up to 70–80% of confluence were washed with PBS and fixed with 10% of fixation solution diluted in deionized water. After 20 min of incubation at 25 °C MSCs were stained with 20 mg/ml X-gal diluted in the staining buffer for 24 h at 37 °C without CO₂. SA- β-gal activity was detected by EVOS Cell Imaging system (Thermo Fisher Scientific, USA).

The positive blue staining of SA-β-gal was used to estimate cellular senescence. The H460 cells (5 × 10³ cells/well) were seeded on 96-well plates and incubated overnight. After being treated with various concentrations (0, 25, and 50 µM) of CTPPU for 48 h, cells were stained with the Senescence-Galactosidase Staining Kit (Cell Signaling Technology), following the same method as described previously [51 (link)]. Five random fields were captured from each well, and the total number of cells and SA-β-gal positive cells were determined using ImageJ software (NIH, Bethesda, MD, USA).

Evaluation of cell aging was carried out to identify the activity of the enzyme SA-β-Galactosidase. Cells (100,000 each) were plated on 3 cm Petri dishes (Corning, USA) and cultivated for 3 days. Then the medium was removed, cells were washed with PBS, and fixed with 4% formaldehyde solution. The staining was carried out using a senescence-galactosidase staining kit (Cell Signaling, Danvers, MA, USA) according to the manufacturer’s instructions. SA-β-Gal activity was detected by cell blue staining visualized under a light microscope.

Cells were seeded in 6-well plates and upon reaching 50–60% confluency, the medium was removed. The cells were washed in PBS before being fixed with the 1 × fixative solution provided with the senescence-galactosidase staining kit (Cell Signaling Technology; #9860). Following the manufacturer’s instructions, a fresh beta-galactosidase staining solution was prepared. After washing with PBS twice, cells in each well were stained with a 1 mL staining solution. After 16-h incubation at 37 °C in a dry incubator, senescent cells were identified for a positive beta-galactosidase-dependent dye conversion.

To visualise senescent-like cells, a Senescence-Galactosidase Staining Kit (Cell Signaling Technology, 9860) was used according to manufacturer’s instruction. The staining was carried out at pH 6.0 in a humidified chamber overnight at 37 °C. Imaging was carried out using an Olympus wide field microscope CK30 10x objective and captured with Dino-Lite adaptor and Dino-Lite software. For SASP experiments, conditioned medium cells was harvested from intoxicated HT1080 cells at 4 days, unless indicated otherwise, and filter-sterilised before incubation with naïve HT1080 cells or non-differentiated THP1s. Cells were incubated for 7 days before assaying DNA damage, infection or SA-β-Galactosidase activity.