Rhod 2 am

Calcium transients were mapped using Rhod-2AM (Thermo Fisher, #R1245MP, Waltham, MA, USA) dye while action potentials were mapped using RH-237 dye. Rhod-2AM (Thermo Fisher, #S1109, Waltham, MA, USA) dye was reconstituted in DMSO to make 1 mg/mL of stock solutions. The stock solution was diluted in Pluronic acid (Gibco, #24040032, Billings, MT, USA) in a 1:1 ratio. The diluted dye was then added to dye-free Tyrode’s solution to obtain a 10 mM solution. Similarly, RH-237 dye was dissolved in DMSO to make 1 mg/mL stock solutions. The dissolved dye was added to Tyrode’s solution at a concentration of 5 mM. For mapping of calcium transients, cells were washed 3 times with warm Tyrode’s solution followed by incubation with the diluted dye (10 mM) for 15 min at 37 °C. Cells were washed 3 times with Tyrode’s solution and then mapped immediately. For mapping of membrane voltage, cells were washed 3 times with dye-free Tyrode’s solution, followed by incubation with 5 mM of RH237 dye for 5 min at 37 °C. Finally, cells were washed 3 times with Tyrode’s solution and mapped immediately. Calcium or voltage signals were mapped using MiCAM03 N256 Single Camera System (Scimedia Ltd., Costa Mesa, CA, USA) and analyzed using BV Workbench (Version 2.7.2, Scimedia Ltd., Costa Mesa, CA, USA).

Cells at a final concentration of 1 × 10⁵ cells/mL were seeded into 6-well plates and subjected to H/R intervention. Fresh kidney tissues were ground, and pre-cooled PBS washed 2 times. Single-cell suspension of 1 × 10⁵ cells/mL was prepared. For detection of intracellular Ca²⁺ concentration, 4 μL Fluo-3 AM (Solarbio, Beijing, China) was added to H/R NRK-52E cells and kidney single-cell suspension, and the Fluo-3 AM final concentration was 5 μM. For detection of mitochondrial Ca²⁺ concentration, 4 μL Rhod-2 AM (Thermo Scientific, MA, USA) was added to H/R NRK-52E cells and kidney single-cell suspension, and the Rhod-2 AM final concentration was 5 μM. For detection Ca²⁺ concentration in ER, 4 μL Mag-Fluo-4 AM (Thermo Scientific) was added to H/R NRK-52E cells, and the Mag-Fluo-4 AM final concentration was 5 μM. Cells were incubated at 37 °C for 30 min and washed twice with PBS. The incubation was continued by adding 2 mL PBS into the incubator at 37 °C for 30 min. Samples were collected, and 500 μL PBS was added to resuspend the cells for fluorescence intensity detection at excitation wavelength of 500 nm and emission wavelength of 525 nm using flow cytometry.