Fluo 4 am assay kit

Intracellular Ca²⁺ concentration was detected using a Fluo-4 AM assay kit (Beyotime Biotechnology Institute, China). The stock of Fluo-4 AM was diluted to 0.5–5 μM with PBS, culture medium was discarded from the cell samples to be tested, and the cells were washed three times using PBS (37°C preheated). After that, 1,000 μL working solution was added to six-well plates and incubated in a constant temperature incubator (37°C) for 45 min. The working solution was discarded, and PBS (37°C preheated) was used to wash the cells three times. Finally, the cells were collected for detection of the concentration of intracellular Ca²⁺ using a fluorescence microscope and a fluorescence microplate reader.

Detection of oxidative stress damage included measurements of ROS, changes in mitochondrial membrane potential, and calcium accumulation. ROS were measured with a dihydroethidium fluorescence probe (Catalog #KGAF019, KeyGen Biotech, Nanjing, China). Mitochondrial membrane potential and calcium accumulation were detected using a JC-1 mitochondrial membrane potential assay kit (Catlog#10009172, Cayman, Ann Arbor, MI, USA) and Fluo-4 AM assay kit (Catalog # S1060, Beyotime Biotechnology, Shanghai, China). All these detection experiments were conducted following the manufacturers’ instructions. Fluorescence changes in the HFL-1 cells were detected, photographed, and counted in at least 5 random fields of each slide with a fluorescence microscope at 200× magnification (Leica, Germany).