Las x 3.5.5.19976 software platform

For the cell imaging, the cells (MCF7: 25,000 cells/cavity; MDA-MB-231: 19,000 cells/cavity; T47D: 38,000 cells/cavity; MCF10A: 6000 cells/cavity) were grown on µ-Slide 8-well plates (ibidi; Gräfelfing, Germany). After 24 h, the medium was renewed, and the cells were incubated in their respective growth media for 72 h. As described above, the cells were loaded with ZP1 in assay buffer with 0.3% BSA for 30 min. After washing the cells twice, the medium was added, and images were taken with a Leica TCS SP8 CLMS equipped with an LAS X 3.5.5.19976 software platform (Leica, Wetzlar, Germany) using an HC PL APO CS2 63×/1.20 water objective. For the excitation, a 488 nm laser was used. The detector was set to a bandwidth of 500–550 nm.

Intracellular free zinc measurement was performed with a slightly modified from a protocol published before [37 (link)]. Briefly, cells were grown in 96-well plate cavities up to 70–75% confluency before loading with 2.5 µM Zinpyr-1 in a loading buffer (10 mM HEPES; pH 7.35; 120 mM NaCl; 5.4 mM KCl; 5 mM glucose; 1.3 mM CaCl₂; 1 mM MgCl₂; 1 mM NaH₂PO₄; 0.3% (w/v) BSA). Next, aerogel swelling supernatans or metal salt solutions (ZnSO₄, ZnO, CaCl₂, AgNO₃) were added and fluorescence was measured after 30 min incubation on a Tecan Infinite M200 reader using excitation/emission wavelengths of 492/527 nm for Zinpyr-1 [38 (link)]. In addition, confocal laser scanning microscopy (Leica TCS SP8 CLMS equipped with LAS X 3.5.5.19976 software platform; Leica, Wetzlar, Germany, using a HC PL APO CS2 63x/1.20 water objective; filters settings were λ_Exc 488 nm/ λ_Em 500–550 nm) was performed to monitor cellular distribution of Zinpyr-1-accessible zinc.