Fluo 4 am

Intracellular Ca²⁺ transients were monitored in ND7-23 wild type neurons using the Ca²⁺ sensitive fluorescent indicator Fluo-4 AM (Biotium Inc., Hayward, CA, USA) prepared as a 1 mM stock solution in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and stored at − 20 °C.
Coverslips with ND7-23 cells were incubated with 4 μM Fluo-4 (diluted in culture medium) for 1 h in a tissue culture incubator. Before recording the Ca²⁺ transients, the coverslips were washed with Tyrode solution to remove extracellular indicator and left to equilibrate in the bath solution for 20 min before the addition of venom (0.3 µg/mL; concentration used in vas deferens electrophysiological experiments). All Ca²⁺ measurements were carried out at room temperature. The images were recorded using a Grasshopper3 USB3 camera model GS3-U3-15S5M-C (FLIR Integrated Imaging Solutions Inc., Richmond, Canada) attached to a Zeiss Axioskop 50 upright epifluorescence microscope (Carl Zeiss, Oberkochen, Germany). Excitation light was provided by a 50 W mercury short arc lamp (Osram, Germany) and filter set 9 (Carl Zeiss) that consisted of an excitation filter (BP 450–490 nm), beam splitter (FT 510 nm) and emission filter (LP 520 nm). Image acquisition was controlled with the software WinFluor software (John Dempster, University of Strathclyde, UK) and obtained at a rate of 2 frames/s and exposure times of 100–500 ms.