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. Author manuscript; available in PMC: 2014 Jun 1.

Published in final edited form as: Cell Microbiol. 2013 Jan 9;15(6):961–976. doi: 10.1111/cmi.12089

Fig. 1 — A. Generation of ROS in primary GECs after treatment with ATP measured using CM-H₂DCFDA (DCF, a dye oxidized to green fluorescent by cellular H₂O₂) staining by cytofluorimetry at 24 h. High fluorescence population (right-shift of the green fluorescence distribution peak) indicates strong production of ROS upon ATP treatment at 24 h.

B. Kinetics of cellular ROS in living cells upon P2X₇ agonist (3 mM ATP), antagonist (5 mM oxATP), and non-specific nucleotide (3 mM ADP) treatments were examined using DCF by fluorescence microscopy imaging combined with DIC during 24 h. The DIC verified the total number of cells in the samples.

C. The mean fluorescence intensities were determined by NIH-Image J analysis for 30 min, 2, 6, and 24 h time periods. The fluorescence intensity was normalized to the level of fluorescence recorded prior to stimulation with ATP. 100 μM H₂O₂ is used as positive control. 10 mM DMSO (diluent for DCF dye) was used to determine background control levels. The results are representative of at least 3 separate experiments performed in duplicate.

Fig. 1 — A. Generation of ROS in primary GECs after treatment with ATP measured using CM-H₂DCFDA (DCF, a dye oxidized to green fluorescent by cellular H₂O₂) staining by cytofluorimetry at 24 h. High fluorescence population (right-shift of the green fluorescence distribution peak) indicates strong production of ROS upon ATP treatment at 24 h.

B. Kinetics of cellular ROS in living cells upon P2X₇ agonist (3 mM ATP), antagonist (5 mM oxATP), and non-specific nucleotide (3 mM ADP) treatments were examined using DCF by fluorescence microscopy imaging combined with DIC during 24 h. The DIC verified the total number of cells in the samples.

C. The mean fluorescence intensities were determined by NIH-Image J analysis for 30 min, 2, 6, and 24 h time periods. The fluorescence intensity was normalized to the level of fluorescence recorded prior to stimulation with ATP. 100 μM H₂O₂ is used as positive control. 10 mM DMSO (diluent for DCF dye) was used to determine background control levels. The results are representative of at least 3 separate experiments performed in duplicate.