Extracellular Nucleotide Quantification

Supernatants of HUVEC and PMNs previously incubated with ABC (20 µmol/L, 37°C) were collected (3 and 8 min and 4 h), mixed with MeOH (10 min, 4°C) and centrifuged (10 min, 15.000 rpm). 100 µL of the respective samples were dried under N₂ flow prior to the addition of H₂O and subsequent analysis of the extracellular nucleotides by means of a 1200 Series uHPLC coupled to a 6495 QqQ/MS (Agilent Technologies) (Chen et al., 2009 (link); Zhang et al., 2014 (link)).
To determine the levels of CBV-TP, supernatants of HUVEC and PMNs previously incubated with ABC (20 µmol/L, 37°C) were collected (at 4 h) and treated with phosphatase to degrade the CBV-TP to CBV, and CBV-TP was measured as total CBV (CBV-TP + CBV) by uHPLC coupled to a 6495 QqQ/MS (Agilent Technologies).