Annexin 5 propidium iodide staining

Cell viability was assessed by annexin V/propidium iodide staining (eBioscience, San Diego, CA, USA). Briefly, cells were harvested with trypsin-EDTA, suspended in staining buffer, stained for 20 min with annexin V and propidium iodide, and analyzed using a BD FACSCalibur TM and BD CellQuest TM Pro (Becton, Dickinson Biosciences, San Jose, CA, USA).
P2X 7 channel uptake YO-PRO-1, a 629-Da fluorescent nucleic acid dye that is transported across the cell membrane by P2X 7 , was used to quantify the uptake of extracellular ATP, as previously described (Cankurtaran-Sayar et al. 2009) . In brief, cells were collected with trypsin-EDTA, centrifuged at 1,500 rpm for 5 min, washed once with cold phosphatebuffered saline, and stained with 2 μM YO-PRO-1 (Invitrogen, Carlsbad, CA, USA) for 30 min in the dark. The mean fluorescence intensity was then analyzed using a BD FACSCalibur TM and BD CellQuest TM Pro (Becton, Dickinson Biosciences, San Jose, CA, USA).