The extracellular nucleotides were detected using SytoxGreen staining [20 (link)] Briefly, isolated from bone marrow cells neutrophils were transferred to the SytoxGreen dissolved in HBSS (PanEco, Moscow, Russia) 1:1000. Neutrophils were incubated in a black 96-well plate (SPL Life Sciences, Pocheon, Korea) in a concentration of 2 × 105 cells per well at 37 °C and 5% CO2 in the presence of 5 µM of A23187 for 3 h. The samples were analyzed using GloMax Microplate reader Multisystem (Promega, Madison, WI, USA) in a fluorescence mode, filter 490 nm.
Neutrophils were also loaded to the coverglass-bottom slides (Ibidi, Gräfelfing, Germany), incubated 3 h in presence or without of 5 µM of A23187, then fixed with 4% paraformaldehyde (Applichem, Darmstadt, Germany). The samples were stained with anti-histone H3 (citrulline R2 + R8 + R17) antibody (Abcam, Boston, MA, USA, ab5103) and the secondary goat-anti-rabbit PE-conjugated (Sigma-Aldrich, St. Louis, MO, USA, P9537). The nuclei were stained with Hoechst (PanEco, Moscow, Russia), dilution 1:1000.
Neutrophils were also loaded to the coverglass-bottom slides (Ibidi, Gräfelfing, Germany), incubated 3 h in presence or without of 5 µM of A23187, then fixed with 4% paraformaldehyde (Applichem, Darmstadt, Germany). The samples were stained with anti-histone H3 (citrulline R2 + R8 + R17) antibody (Abcam, Boston, MA, USA, ab5103) and the secondary goat-anti-rabbit PE-conjugated (Sigma-Aldrich, St. Louis, MO, USA, P9537). The nuclei were stained with Hoechst (PanEco, Moscow, Russia), dilution 1:1000.
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