Fluo 3 am

Intracellular Ca²⁺ levels were quantified using a Ca²⁺ quantification kit (Abcam, ab112115) following the standard manufacturer’s protocol. Fluorescence signals were detected using a microplate spectrophotometer at Ex/Em = 540/590 nm (Varioskan LUX, Thermo). Cytosolic Ca²⁺ levels were also measured by flow cytometric estimation of Fluo-3 AM. The cells were collected and loaded with 3 μM Fluo-3 AM (Abcam, ab145254) for 1 h at 37 °C in the dark, and then resuspended with 500 μl phosphate-buffered saline. The fluorescence signal was recorded using a flow cytometer at Ex/Em = 488/525 nm and analyzed by NovoExpress software (NovoCyte, ACEA Biosciences, San Diego, CA, USA).

Coomassie Brilliant Blue (CBB) kit (A045-2-2) and detection kits for total antioxidant capacity (T-AOC) (A015-2-1), glutathione peroxidase (GSH-PX) (A005-1-2), nitric oxide (NO) (A013-2-1), nitric oxide synthesis (NOS) (A014-2-2), malondialdehyde (MDA) (A003-4-1), lipid peroxide (LPO) (A106-1-3), and protein carbonyl (Carbonyl) (A087-1-2) were the products of Nanjing Jiancheng Institute of Bioengineering and Technology (Nanjing, China); detection kits for glutathione (GSH), glutathione disulfide (GSSG) (G263), and DNA damage quantification kit (DK02) were the products of Dojindo Molecular Technologies (Rockville, MD, USA); assay kits for reactive oxygen species (ROS) and superoxide dismutase (SOD) (ab139476), genomic DNA extraction kit (ab156900), and intracellular calcium ion ([Ca²⁺]i) detection kit (Fluo-3/AM) (ab145254) were the products of Abcam (Cambridge, UK); apoptosis detection kit (556547) was the product of BD Biosciences (San Jose, CA, USA); mitochondrial membrane potential (MMP) detection kit (JC-1) (M8650) was the product of Solarbio Co. (Beijing, China); RNAiso™ Plus kit (9108) and PrimeScript™ RT reagent kit (6210A) were the products of Takara (Dalian, China).