Pluronic f 127

To assess the intracellular amount of potassium, P815 cells were first incubated for different time points with 100 pmol/mL β-estradiol and serum-free control. Cells from each treatment condition were seeded into 96-well plates (30000 cells/well, Bioland Scientific, USA) in quintuplicate and wells were loaded with 10 µM of the cell permeant potassium-binding benzofuran-isophthalate acetoxymethyl ester (PBFI-AM, fluorescent K binding probe, ab142804, Abcam) with 0.1% Pluronic F-127 (Yeasen Biotech, Shanghai, China). After incubation for 2 h at 37 °C, excess PBFI-AM was removed using PBS. P815 were plated and signal intensity was measured with Varioskan Flash (Thermo Scientific). Excitation ratios of fluorescence at 340 and 380 nm, measured at emission 500 nm were used to detemine the intracellular content of K⁺, according to the manufacturer-suggested protocol. Measurements were corrected for autofluorescence values and presented as percent of untreated control.

The cardiomyocytes were plated on a 35 mm confocal dish loaded with 4 μM Fluo-4 AM (Yeasen, China) and incubated at 37 °C for 20 min in PBS (Servicebio, China) containing 0.04% Pluronic F-127 (Yeasen, China). PBS was changed to the Cardiomyocytes Maintenance Medium (Cellapy, China). Loaded samples were transferred under a TCS‐SP5‐RS confocal microscope (Leica, Germany). Laser emission at 488 nm was used for stimulation and emitted fluorescence at 530 nm was acquired. Samples were then stimulated with freshly prepared solution of caffeine (20 mM) and emitted fluorescence acquired to record transient alteration in cytosolic calcium levels.

Briefly, HepAD38 cells were seeded in 96-well plates at 2 × 10⁴ cells per well and cultured in DMEM/F12 for 24 h. Cells were then washed with HBSS buffer three times and incubated with 5 μM Fluo4-AM containing 0.05% PluronicF-127 (Yeasen Biotech, Co., Ltd.) at 37°C for 1 h. The cells were washed three times with HBSS and incubated at 37°C for another 30 min to make the fluorescence probe completely de-esterifying. The cells were then incubated with serially diluted compounds and the Ca²⁺ levels were determined based on the fluorescence (excitation wavelength 480 nm, emission wavelength 525 nm) measured using a Perkin Elmer Envision Multi-label Plate Reader.