Ca2 quantification kit

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).

Intracellular Ca²⁺ levels were quantified using a Ca²⁺ quantification kit (Abcam, ab112115) following the standard manufacturer’s protocol. Fluorescence was determined using a microplate spectrophotometer at Ex/Em = 540/590 nm (Varioskan LUX, Thermo). Additionally, cytosolic Ca²⁺ levels were measured by flow cytometric estimation of Fluo-4 AM. The cells were collected and loaded with 5 μM Fluo-4 (Beyotime, ab145254) for 30 min at 37° C, and then resuspended in 500 μL of phosphate-buffered saline. Fluorescence signals were recorded with a flow cytometer at Ex/Em =488/525 nm and analyzed with NovoExpress software (NovoCyte, ACEA Biosciences, San Diego, CA, USA).