Calcium Signaling Dynamics in Cells

Cells were incubated with the labelled calcium indicator Fluo-4AM (ThermoFisher Scientific, F14201) in culture medium for 45 min (10 µM, 37 °C). Cell were incubated for 5 min (37 °C) to allow for de-esterification of AM esters. For baseline measurements of calcium signalling, artificial CSF (aCSF) was perfused over the cells for 5 min (see Table 2). 5HT (10 µM in dH₂O, 14927) was perfused for 5-minutes suspended in aCSF before a 5-min aCSF washout period. A minimum time-course of 3-min has been used in previous research to investigate 5HT induced calcium dynamics at similar compound concentrations [12 (link), 13 (link)]. Imaging was performed using a fluorescence microscope (Nikon Eclipse FN1) with a 20× objective. Fluo-4AM fluorescence was excited at 488 nm and captured every 2 s to create a time-lapse (Hamamatsu Orca Flash V2). The fluorescence was calculated from five regions of interest containing approximately 3–10 cells using Fiji software (ImageJ, NIH).Table 2

aCSF constituent used for perfusion over cells for baseline calcium measurements and as a vehicle for serotonin.

Component	Final concentration (mM)
NaCl	126
KCL	2.5
NaHCO3	26
KH2PO4	1.25
MgSO4	1
CaCl2	2
Glucose	10

pH 7.3–Continuous bubbling with CO₂ throughout the experiment.