Ion potassium green 2 am

Cells were loaded with a potassium indicator, ION Potassium Green-2 AM (ab142806, Abcam, UK) at 5μM for 30 min at room temperature, according to the manufacturer’s instructions. After two washes in warmed PBS, cells were imaged using an Olympus FV3000 inverted confocal microscope. The fluorescence signal between 530nm and 560 nm was recorded.

Fluo-4 AM (Molecular Probes) and ION Potassium Green-2 AM (APG-2 AM, Abcam) are used to detect the concentration of Ca²⁺and K⁺, respectively, according to the published methods^{108 (link),109 (link)}. For Ca²⁺ measurements, the treated C. elegans were loaded with 100 µM Fluo-4 AM for 1 h in M9 buffer. C. elegans were washed >3 times and then photographed by Olympus BX63 epifluorescence microscope. For K⁺ measurements, treated C. elegans were labeled in 5 μM APG-2 AM in each buffer of different potassium concentrations for 1 h. The labeled C. elegans were cultured in the corresponding buffer with added OP50 for 1 h to remove the free dye in the intestine. The washed C. elegans were photographed under the same conditions. The excitation and emission wavelengths of Fluo-4 AM were 489 and 508 nm. For APG-2 AM, they are 526 and 550 nm, respectively. At least three repeats were performed for each condition and at least 20 animals were measured per treatment. The average optical density of each worm was measured using Image Pro Plus v6.0.

Briefly, the embryos were loaded with 5 μM ION Potassium Green-2 AM (Abcam) at 37°C for 30 min according to a previous report [18 (link)]. The embryos were washed three times with KSOM, and the intracellular fluorescence intensity was measured. The excitation wavelength was set to 526 nm (6-JOE), and emission was detected at 540–560 nm (Fig 2A). The same experiments were repeated at least three times.
In total, 180 mouse embryos were observed in this study. For Na⁺ imaging, 96 embryos were studied, including 55 in the control group [morula (n = 20), blastocyst (n = 18), and hatching (n = 17)] and 41 in the ouabain group [morula (n = 17), blastocyst (n = 12), and hatching (n = 12)]. For K⁺ imaging, 84 embryos were used, including 50 in the control group [morula (n = 22), blastocyst (n = 11), and hatching (n = 17)] and 34 in the ouabain group [morula (n = 16), blastocyst (n = 9), and hatching (n = 9)].