Fluo 4 am kit

Ca²⁺ concentration was detected by Fluo-4 AM kit (S1060, Beyotime Biotechnology, China) according to the manufacturer’s instructions. About 10,000 cells were obtained by an FACS Calibur system (BD Biosciences, New Jersey, BD, USA) for analysis under FlowJo software (version 10.0.7).

Cells were seeded in thin-bottom dishes at a density of 1 × 10⁴ cells per dish. After fixation in 4% paraformaldehyde for 10–15 min, the cells were washed three times with PBS, stained according to the instructions of a Fluo-4 AM kit (Beyotime Biotechnology, China), and incubated at 37°C for 30 min for fluorescent probe loading. Subsequently, the cells were washed three times with PBS. After further incubation for 30 min, the nuclei were stained with DAPI (Solarbio Science & Technology, China). An anti-fluorescence quencher was added dropwise. Photographs were taken with a laser confocal microscope. ImageJ was employed to analyze and measure the mean fluorescence intensity.

The Fluo-4 AM Kit (S1060, Beyotime, China) was applied to evaluate intracellular Ca²⁺ concentration. This experimental procedure refers to the previous study [26 (link)]. NRK-52E cells were subjected to multiple rinses and then probed with PBS-diluted Fluo-4 AM at 37°C. After 30 min, a laser scanning confocal microscopy (FV500-IX71, Olympus, Japan) was used to monitor Fluo-4 fluorescence excited at 494 nm. The value of fluorescent intensity was recorded to calculate the concentration ([Ca²⁺]_i).