Fura 2 am

The metal ions in A. flavus were assayed by staining with the Ca²⁺-specific fluorescence dye Fura-2-AM (Fura-2 acetoxymethyl ester, 40702ES50, Yeasen, Shanghai, China), Na⁺-specific fluorescence dye SBFI-AM (sodium-binding benzofuran isophthalate acetoxymethyl ester, GC44876, GLPBIO, Montclair, CA, USA) and K⁺-specific fluorescence dye PBFI-AM (potassium-binding benzofuran isophthalate acetoxymethyl ester, GC18477, GLPBIO, Montclair, CA, USA) as previous reports [36–39 ]. All A. flavus strains were cultured in YES media, then collected and washed three times with PBS buffer to remove the media. The hyphal filaments were resuspended in PBS buffer containing 10 mM fluorescence dyes (Fura-2-AM, SBFI-AM, or PBFI-AM) and incubated at 37°C for 20–30 min. After washing three times with PBS buffer, the fluorescence intensities from the different stained hyphal filaments were measured at excitation wavelengths of 340/380 nm and an emission wavelength of 505 nm by using a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski, VT). The relative metal ions concentrations in different hyphal filaments were calculated based on the ratio of fluorescence intensity after dual-wavelength excitation. The unstained strains were used as controls. Each treatment was performed with three independent biological replicates. Statistical comparisons were performed by Student’s t-test.

4T1 cells were seeded into a 96-well plate and stained with 2 μM Fura-2 AM and 0.05% Pluronic F127 (Yeasen Biotechnology, China) for 30 min in an incubator according to the manufacturer’s guidelines. Cells were washed two times in HEPES. Cells were then put into the spectrophotometer which has its heating elements. The cells were subjected to heat treatment at 43°C for 1 h and the intracellular calcium flux was detected dynamically at 2 min intervals. Changes in intracellular Ca²⁺ concentrations were expressed as ratios of Fura-2 fluorescence with excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. The fluorescent intensity of Fura‐2 was measured by a dual‐excitation wavelength method (340/380 nm) with the spectrophotometer.

Calcium concentration was detected as previously reported [33 (link)]. Briefly, BV2 cells were incubated with 4 μM Fura-2/AM (YEASEN, Shanghai, China, 40702ES50) for 30min, and captured the fluorescence intensities when excited at 340 and 380 nm and emitted at 510 nm. The calcium concentration was calculated as followed: the fluorescence intensities at 340 nm/ the fluorescence intensities at 380 nm.