Chromatographic analysis was performed with an HPLC System (Beckman Gold 126/166NM, Beckman Coulter) equipped with a reverse-phase column (Synergi Fusion C18, 5 μm; 150 × 4.5 mm, Phenomenex). The nucleotides and nucleosides were separated using a pH 5.1 mobile-phase buffer (0.125 M citric acid and 0.025 M KH2PO4) along with 8% acetonitrile (Sigma Aldrich) over 10 min at a flow rate of 0.8 mL/min. UV absorption spectra were measured at 254 nm. HPLC-grade standards used to calibrate the signals were dissolved in AIM V serum-free medium (Invitrogen, Paisley, UK), pH 7.4, 0.2 μm sterile-filtered and injected in a buffer volume of 20 μL. The retention times (Rt, in min) of standards were: AMP, 2.15; NAD+, 2.8; ADPR, 3.2; and ADO; 5.56. Peak integration was performed using a Karat software (Beckman Coulter).
ACN-treated melanoma cell supernatants (see above) were evaporated by speed-vac, reconstituted in mobile-phase buffer, and assayed by HPLC.
The qualitative identity of HPLC peaks was confirmed by co-migration of known reference standards. The presence of ADO was also confirmed by spiking standard (50 μM ADO), followed by chromatography. Quantitative measurements were inferred by comparing the peak area of samples with calibration curves for peak areas of each standard compound. Product concentrations were expressed as nmol/30 min/number of cells (5×105 cells).
ACN-treated melanoma cell supernatants (see above) were evaporated by speed-vac, reconstituted in mobile-phase buffer, and assayed by HPLC.
The qualitative identity of HPLC peaks was confirmed by co-migration of known reference standards. The presence of ADO was also confirmed by spiking standard (50 μM ADO), followed by chromatography. Quantitative measurements were inferred by comparing the peak area of samples with calibration curves for peak areas of each standard compound. Product concentrations were expressed as nmol/30 min/number of cells (5×105 cells).