Vti hcs microscope

MCCs or peripheral neuron precursors were seeded in 96-well plates and differentiated as described above. Before measuring the changes in concentration of intracellular free Ca²⁺ ([Ca²⁺]_i), differentiation medium was changed to artificial cerebrospinal fluid (aCSF): NaCl [140 mM], KCl [3 mM], CaCl₂ [2.5 mM], MgCl₂ [1 mM], Na₂HPO₄ [1.2 mM], pH 7.4, and the cells were loaded with Fluo-4 Direct™ Calcium Assay Kit and H-33342 for 30 min at 37 °C. For experiments requiring a pre-incubation phase, loading with Fluo-4 was performed in parallel. The changes in [Ca²⁺]_i were monitored with a VTI HCS microscope (ThermoFisher Scientific, Pittsburgh, USA) equipped with an incubation chamber providing an atmosphere with 5% CO₂ at 37 °C. Test compounds were administered by an automated pipettor at 10 s after the first picture was taken. Images were taken as fast as possible for 45 s (approx. one image/second) and exported as .avi video files. The video files were afterwards analysed with the CaFFEE software (Karreman et al. 2020 (link)) to obtain single cell time-course information on [Ca²⁺]_i. The cell densities and number of evaluated cells were very similar for all experimental conditions. However, the overall cell numbers shown in some figures may differ, because different numbers of experiments were included in the evaluation.

Sensory neurons were cultured in 96-well plates after thawing. Cells were loaded with the Ca²⁺-indicator Fluo-4 (Thermo Fisher Scientific). Monitoring of [Ca²⁺]_i was performed using a VTI HCS microscope (Thermo Fisher Scientific) equipped with an automated pipettor and an incubation chamber providing an atmosphere with 5% CO₂ and 37 °C. Cells were imaged for 45 s. Test compounds were automatically applied after baseline recording (10 s). The images were exported as *.avi video files and analyzed with the CaFFEE software. Details are given in a dedicated technology paper^{41 (link)} and in Supplementary Material.