96 well cellular senescence assay kit

SA-beta-gal activity was measured with the 96-well cellular senescence assay kit (Cell Biolabs, San Diego, CA). With few exceptions, cell culture and treatment procedures were similar to polycaspase assay procedures. SA-beta-gal activity was measured at 0.5, 1 and 2H. Floating and adherent cells collected after treatment were washed with phosphate buffered saline (PBS), pooled, suspended in 400 μL cell lysis buffer (1x), and kept on ice for 10min. Then, cell suspension was dissolved by vortex. From this, 100 μL solution was frozen at -80°C for about 1 h and used it for CyQuant cell proliferation assay. From the remaining solution, 200μL was mixed with an equal amount of 2x reaction buffer containing SA-beta-gal substrate and incubated at 37°C for 1 h in the dark. After incubation, the solution was mixed by vortex, and a 200-μL solution was mixed with 800μL stop solution. From this, a 200-μL solution was used to determine SA-beta-gal activity in 96-well flat black-bottom plates. Fluorescence was measured in the microplate reader at 360/465 nm (ex/em), and RFU values were normalized to those obtained from CyQuant cell proliferation assay.

The qualitative activity assay is measured according to the manufacturer’s instructions (Cell Signaling Technology) using X-gal (5-bromo-4-chloro- 3-indolyl β-D-galactoside) staining at pH 6.0. Blue stained cells are visualized by light microscope.
For the quantitation of the senescence associated β-Galactosidase activity we used a modified assay, which is described elsewhere [17 (link)]. Briefly, we used the 96-Well Cellular Senescence Assay Kit (Cell Biolabs, Inc.). Here, the activtiy is measured by the rate of conversion of 4-methylumbelliferyl-α-D-galactopyranoside to a fluorescent hydrolysis product 4-methylumbelliferone at pH 6.0. Cells were grown before in 60-mm plates for 1 day (high cell passages) or until confluence (low cell passage). The lysed cells were then used for the β-Galactosidase activity assay and for measuring the DNA concentration by Quant-iT™ PicoGreen™ dsDNA Assay Kit (Thermo-Fisher). The β-Galactosidase activity reactions were carried out at 37 °C for 1 h. The reaction mixture was read by using a 96-well plate using a plate reader with excitation at 385 nm, emission at 465 nm. The relative β-Galactosidase activity is expressed as: [fluorescence of β-Galactosidase activity] divided by [fluorescence of DNA concentration]. Results were calibrated to the relative activity of DFC_S (relative units).

Human HSC LX2 was received as a kind gift from Prof. Scott Friedman. Human fetal hepatocytes LO2 was received as a kind gift from A/Prof. Victor Yu. MgIG was obtained from Jiangsu Chia-Tai Tianqing Pharmaceutical Co., Ltd. (Nanjing, China). Compound structure and detailed information is supplied in Supplementary Figure S1. PCR primers were purchased from Integrated DNA Technologies (Coralville, IA, United States). The following antibodies were used: Anti-collagen-1 was purchased from Abcam (Cambridge, United Kingdom), anti-αSMA from Agilent Dako (Santa Clara, CA, United States); anti-GAPDH, anti-phospho-ERK, anti-ERK, anti-phospho-Akt, anti-Akt, anti-phospho-JNK, anti-JNK, anti-SMAD2/3, anti-SMAD4, secondary anti-mouse and anti-rabbit were purchased from Cell Signaling Technology (Danvers, MA, United States); anti-phospho-p38, anti-p38 and anti-p27 were purchased from Santa Cruz Biotechnology (Dallas, TX, United States). 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) was purchased from Duchefa Biochemie (Haarlem, Netherlands) and propidium iodide (PI) was purchased from Sigma-Aldrich (St. Louis, MO, United States). 96-well cellular senescence assay kit was purchased from Cell Biolabs (San Diego, CA, United States).