Vivaspin 500 membranes

T84 EV and ENPP1-OE were plated on 5-cm² inserts (Corning) and grown to resistance (>1000 ohms^.cm²). The inserts were then washed, equilibrated in HBSS+, and treated apically and basolaterally with 300 µM Ap3A (Sigma-Aldrich). Aliquots of equivalent volumes were sampled both apically and basolaterally at 5, 20, 60, and 120 min. The samples were flash frozen in liquid nitrogen and stored at –80°C until analyzed by HPLC.
Purines were quantified as described elsewhere (Lee et al., 2018 (link)). Briefly, analyses were performed on an Agilent Technologies 1260 Infinity HPLC using a C18 column (100 Å, 150 × 4.6 mm with mobile phase A: 50 mM KH₂PO₄, 5 mM tetrabutylammonium bisulfate, pH 6.25; mobile phase B: acetonitrile; column temperature: 30°C; flow rate: 1 ml/min; 75 µl injection). Samples were filtered through VIVASPIN 500 membranes (Sartorius Stedim Biotech, 5000 MWCO, PES) prior to HPLC analysis. Chromatographic separation of the metabolites was performed using a combination of isocratic and gradient methods including column washing and equilibration periods at the end (0 min: 100% A; 7 min: 100% A; 10 min: 97% A; 18 min: 97% A; 45 min: 86% A; 60 min: 50% A; 80 min: 50% A; 90 min: 100% A; 135 min: 100% A). The metabolites were detected by absorption at 254 nm, with their absorbance spectra and retention times verified by coinjection with authentic standards.

Whole murine hearts were collected in 1 mL of 80% MeOH and flash-frozen under liquid nitrogen and stored at −80°C. Adenosine was extracted and quantified in the tissue as described (Lee et al., 2018 (link)). Analyses were performed on an Agilent Technologies 1260 Infinity HPLC using a phenomenex Luna C18(2) column (100 Å, 150 X 4.6 mm) (mobile phase A: 50 mM KH2PO4, 5 mM tetrabuty-lammonium bisulfate, pH 6.25; mobile phase B: acetonitrile; column temperature: 30°C; flow rate: 1 mL/min; 75 μL injection). Samples were filtered through VIVASPIN 500 membranes (Sartorius Stedim Biotech, 5,000 MWCO, PES) prior to HPLC analysis. Chromatographic separation of the metabolites was performed using a combination of isocratic and gradient methods including column washing and equilibration periods at the end (0 min: 100% A; 7 min: 100% A; 10 min: 97% A; 18 min: 97% A; 45 min: 86% A; 60 min: 50% A; 80 min: 50% A; 90 min: 100% A; 135 min: 100% A). Adenosine was detected by absorption at 254 nm, and the absorbance spectra and retention time verified by co-injection with an authentic standard.