4.7. Fura-2AM-Based Quench Assay

Fura-2AM-based quench assay was used to determined TRPM7 function [32]. Cells (5–6 × 10⁴ cells/well) were plated in 96-well plates. The culture medium was completely removed and replaced with fura-2AM loading-buffer (2 mM fura-2AM in BS). Following incubation (60 min at 37 °C), the loading buffer was removed, and the cells were washed once with BS before the addition of fresh BS as the assay buffer. The plates were then transferred to a 37 °C pre-warmed fluorescence plate reader (PerkinElmer Victor X3, San Jose, CA, USA). In the quench assay, the Ca²⁺-independent fluorescence of fura-2AM (excitation 360 nm; emission 510 nm) was monitored by the microplate photometer. After dye loading, cells were incubated with drugs for 5 min. After recording baseline fluorescence for 10 s, 10 mM MnCl₂ was added and the fluorescence was recorded for 300 s. Fura-2 fluorescence decreases as Mn²⁺ enters the cells through TRPM7, displaces Ca²⁺ from the dye, and quenches the fluorescence. Each experiment was repeated three times.