Cellular senescence detection kit spider βgal

MSCs were seeded in confocal dishes. SA-β-gal staining of MSCs was performed with a Cellular Senescence Detection Kit-SPiDER-βGal (Dojindo, Shanghai, China) according to the manufacturer’s instructions. First, MSCs were treated with bafilomycin A1 for 1 h to block the activity of endogenous β-galactosidase. Then, cells were incubated with SPIDER-βGal working solution for 30 min in 37 °C, 5% CO₂. After that, cells were co-stained with anti-p21 antibody (1:200, Proteintech, 27,296–1-AP). Thereafter, the dishes were viewed under a fluorescence microscope, and images were obtained using a Leica DMi8. SA-β-gal-positive and p21-positive cells were analyzed with ImageJ software 1.4.

NHLF or SAEC were seeded at the density of 5 × 10³ cells per well in 96-well plates. After 24-hour incubation, the medium was replaced with fresh complete DMEM, and the cells were treated with increasing concentrations of rGDF15 (62.5–500 ng/mL). After 1-hour incubation, a final concentration of 3 μM etoposide or the vehicle control (DMSO, 0.006% v/v) was added to the cells, and they were incubated for 24 hours. For RNA extraction using RNeasy 96 Kit (Qiagen), the cells were harvested by removing the medium and adding 100 μL of freshly prepared lysis buffer per well. The plates for immunostaining were fixed with 50 μL of 4% formaldehyde (VWR, 9713.1000) per well. After 15 minutes of incubation, the cells were washed 3 times with PBS and then sealed and stored at 4°C until staining. Prior to the SA–β-gal staining, the culture medium was discarded and the cells were washed with fresh cell culture medium. Then the cells were costained using Cellular Senescence Detection Kit–SPiDER-βGal (Dojindo, SG03-10) and Hoechst 33342 for nuclei detection. Next, the cells were directly imaged using ImageXpress Micro system, and the percentage of SA–β-gal⁺ cells was calculated using MetaXpress High Content Image Analysis Software.