Hanks balanced salt solution with ca2 mg2

Changes in [Ca²⁺]_mito were detected through incubation for 30 min, at 37°C in the dark, with the Ca²⁺ probe Rhod-2 AM (Invitrogen) at a final concentration of 4 µmol/L. After incubation, the cells were washed twice with Hanks' Balanced Salt Solution (with Ca²⁺&Mg²⁺) (Beyotime Institute of Biotechnology) to remove the probes, and were analyzed using laser confocal microscopy. Fluorescence images were captured using a laser confocal system mounted on an inverted microscope equipped with an argon–krypton laser. Rhod-2 AM fluorescence (excitation at 559 nm) was detected at a wavelength of 577 nm.[21] (link) In this glucose dependent experiment, the cells were pre-washed for 30 min with low glucose (5.56 mmol/L) containing HBSS, followed by HG (35 mmol/L) containing HBSS. The specific [Ca²⁺]_mito line-scanning method was performed over a single horizontal position consisting of 160 lines at 5 s intervals. The Ca²⁺ level was expressed as a pseudo-ratio value (F/F₀) of the actual fluorescence intensity (F) divided by the basal intensity of the [Ca²⁺]_mito at rest (F₀). F₀ was calculated as the average value obtained during the 50–60 s prior to stimulus application.[22] (link)