2.4. Neurotransmitter Release Quantification

The acetylcholine neurotransmitter, released in cell medium was quantified using fluorometric kit (Acetylcholine, Abcam, Waltham, MA, USA, ab65345). The four neurotransmitters were quantified in supernatant of 14 days differentiated PC12 cells harvested in conditioned medium after addition of Carbachol (1.5 mM) in the medium.

Mass spectrometry was then performed. The cell secretion media were collected. Cells were recovered in 50 µL of H₂O containing 0.1 mM ascorbic acid. Cells were sonicated (4 × 10 s, 100 W; Fisher Scientific, Waltham, MA, USA; Model 505 Sonic Dismembrator). After centrifugation (20,000× g, 30 min, 4 °C), supernatants were collected and protein concentrations were accessed using the Protein Assay kit (Bio-Rad). An isotopic dilution approach was used for the absolute quantification. Either 20 µL of the cell extracts or 50 µL of the secretion medium were mixed with 10 µL of internal standards containing 20 pM of D4-Dopamine, C6-Noradrenalin, D6-Adrenalin, L-DOPA in 0.1 mM ascorbic acid1. Then, 40 µL of borate buffer and 10 µL AccQtag Ultra reagent (AccQ-Tag Ultra derivatization kit, Waters, Guyancourt, France) were added to cell extracts and secretion media. The mixture was incubated 10 min at 55 °C under agitation. Then, 500 µL of ice-cold acetonitrile (ACN) were added and samples were centrifuged (20,000× g, 30 min, 4 °C). The resulting supernatants were dried under vacuum and suspended in 20 µL of H₂O containing 0.1% formic acid (v/v).

Analyses were performed on a Dionex Ultimate 3000 HPLC system (Thermo Scientific, Waltham, MA, USA) coupled with an Endura triple quadrupole mass spectrometer (Thermo Electron, Waltham, MA, USA). The system was controlled by Xcalibur v. 2.0 software (Thermo Electron). Next, 5 µL of samples were loaded into reverse phase Zorbax column (#863600-902; 1 mm × 150 mm, 3.5 µm, Agilent Technologies, Santa Clara, CA, USA, SB-C18). Elution of the compounds was performed at a flow rate of 90 µL/min, at 40 °C (see Supplemental Table S1 for conditions). Buffer A corresponded to H₂O 98.9%/ACN 1%/formic acid 0.1% (v/v/v) and buffer B was ACN 99.9%/formic acid 0.1% (v/v). Dopamine, adrenalin and noradrenalin are measured using the multiple reaction monitoring mode (MRM) according to the settings. The targeted compounds are detailed in Supplement Tables S2 and S3. The selection of the monitored transitions and the optimization of the collision energy (CE) were manually determined. The identification of the compounds was based on precursor ions, daughter ions and retention times obtained for dopamine, adrenalin and noradrenalin and their corresponding internal standards. Amounts of neurotransmitters were quantified according to the isotopic dilution method [26].