Cellular senescence assay kit

Senescence-associated β-galactosidase (SA-β-gal) activity was detected using the cellular senescence assay kit (Chemicon, Temecula, CA), according to the manufacturer’s instructions and as previously described [25 (link)]. In brief, the cells were transfected with anti-miRNA, anti-miR-135b or anti-miR-135a for 48 h and then seeded in seeded in 6-well plates, and incubated for 16 h at 37 °C. Representative microscopic fields were photographed.

The “Chemicon International” Cellular Senescence Assay Kit was used on various passages of growing HPAE cells to qualify their percentage of senescent cells. Senescent-associated β-galactosidase (SA-β-gal) is only present in senescent cells, and Chemicon’s kit provides all reagents needed to detect SA-β-gal activity at pH 6.0 in cell cultures. SA-β-gal catalyzes the hydrolysis of X-gal, causing the accumulation of the blue dye in senescent cells. Detection and quantification of β-gal-positive cells were based on the protocol provided in SA-b-gal-positive cell detection kit (Chemicon’s kit) and a previously established method and approach [86 (link)]. Briefly, the cells were examined on a light microscope with an attached camera using a 10× objective. The β-gal-positive cells were blue. The fraction of β-gal-positive cells was determined by counting the number of blue cells in 5 non-overlapping images from various fields of view within each well. Cells were dislodged by trypsin-EDTA and counted to obtain total number of cells per condition. The β-gal-positive (blue) cell count was divided by the number of total cell count to calculate the percentage senescent cells. Representative images were used for figures.

The HPAECs, EGM, FBS growth supplement, HEPES, 0.25% Trypsin-EDTA, and trypsin-neutralizing solution were used as supplied by Clonetics (San Diego, CA, USA). The HK and PK were purchased from Enzyme Research Laboratory (South Bend, IN, USA). S2302 was purchased from DiaPharma (Franklin, OH, USA). The primary antibody, Goat Anti-Human PRCP, and the secondary Anti-Goat IgG: Whole Molecule, Peroxidase Conjugate were purchased from Bioscience (Long Beach, CA, USA). Mouse Anti-Human β-Actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The “Nitrate/Nitrite Fluorometric Assay Kit” was purchased from Cayman Chemicals (Ann Arbor, MI, USA). The Cellular Senescence Assay Kit was purchased from Chemicon International (Temecula, CA, USA). TRIzol, Super Signal West Femto Maximum Sensitivity Substrate, RiPA Buffer, Super Signal West Femto Maximum Sensitivity Substrate were supplied by ThermoFisher Scientific (Rockford, IL, USA). The Precision Plus Protein standard (Dual Color) was purchased from Bio-Rad (Rockford, IL, USA). Ethidium bromide was from Sigma-Aldrich (St. Louis, MO, USA).