Quantiva

Equal aliquots of chlorophyll a were added to each aqueous medium, and absorption spectra were measured using a UV/VIS spectrophotometer (Genesys 10S; Thermo Fisher Scientific, Waltham, MA, USA). All absorption spectra of chlorophyll a in each medium with continuous illumination treatment were scanned in a range of 250 to 800 nm.
Mass spectrometry experiments were performed on the chlorophyll extracts using the full mass scan mode of a triple quadrupole mass spectrometer (Quantiva, Thermo Fisher Scientific, Waltham, MA, USA) in positive-ion mode. An amount of chlorophyll a extracts (100 µL) in three different media, i.e., EtOH, DW, and C-PC medium, was further diluted using 900 µL methanol solvent with 1% formic acid, and then the mixtures were subjected to ionization by direct-infusion atmospheric pressure chemical ionization (APCI). The following mass spectrometric parameters were used in the experiments: Flow rate 20 μL/min; sheath gas (N₂) pressure 25 psi; auxiliary gas (N₂) pressure 5 psi; ion-transfer tube temperature 150 °C; vaporizer temperature 450 °C; and positive ion discharge current 4 μA. Xcalibur^TM v.4.1 software was used for data acquisition and processing.

The ability of different populations of cells to metabolize ATP to ADP, AMP and adenosine (and from AMP to adenosine) was measured using an ATP and AMP hydrolysis Mass Spectrometry protocol^{69 (link)}. Briefly, cells of interest were isolated by either MACS or flow sorting and then separately cultured at 2 × 10⁴ cells/well in triplicate in 96-well plates at 37 °C in 5% CO₂ for 1 h, in the presence or absence of 50 μM ATP or AMP. Cells were pelleted and supernatants flash frozen in liquid nitrogen and stored at −80 °C. Wells containing no cells and wells containing cells with no added ATP/AMP were used as controls. Supernatants were thawed and metabolites extracted using 2:1 methanol/chloroform followed by 1:1 water and chloroform. Nucleotides were measured by Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS) using a Thermo Quantiva interfaced with a Ultra High performance Liquid Chromatography (UHPLC) Vanquish system (Thermo Scientific, Hemel Hempstead, UK). For chromatography on the UHPLC system, the strong mobile phase (A) was 100 mM ammonium acetate, the weak mobile phase was acetonitrile (B) and the LC column used was the ZIC-HILIC column from Sequant (100 × 2.1 mm, 5 µm). Relative abundance of each metabolite was measured as area under the curve at relevant masses and normalized to stable isotope labelled standards.