Quantifying ATP Release and Dye Uptake in Bone Marrow-Derived Neutrophils

For ATP release assay, BMDNs were left to equilibrate in Tyrode’s buffer (124 mM NaCl, 2.4 mM KCl, 10.8 mM NaHCO₃, 0.4 mM NaH₂PO₄*H₂O, 0.9 mM MgCl₂*6H₂O, 1.8 mM CaCl₂*6H₂O, pH 7.35). Panx1 channel mediated ATP release was activated by reducing osmolarity of the Tyrode’s buffer by decreasing NaCl to 30.24 mM. WT and Panx1^−/− BMDNs were stimulated with appropriate controls for 5 min. Cells were centrifuged at 1200 rpm for 5 minutes and supernatants were collected for ATP measurement using the ATP bioluminescent assay kit (#FLAA, Sigma Aldrich), according to the manufacturer’s instructions.
For dye uptake assay, BMDNs were seeded in 384-well plate and maintained in a physiological solution (136 mM NaCl, 4 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, and 2.5 mM glucose, buffered to pH 7.4 with 10 mM HEPES-NaOH solution). YO-PRO-1 Iodide (5 µM, ThermoFisher Scientific, #Y3603) was added to equilibrate cells. Hypo-osmotic shock was induced to maximally open Panx1 channels. Inhibitors of Panx1 BB-FCF dye-Erioglaucine disodium salt (Sigma Aldrich, #80717)^{24 (link),25 (link)} and spironolactone (Sigma Aldrich, #S3378)^{39 (link)} were prepared in DMSO and used at 1μM and 100 μM, respectively. Global quantification of YO-PRO-1 dye uptake per well was performed using the FDSS/µCELL Functional Drug Screening System (Hamamatsu Photonics).