TLR stimulation results in ATP release from macrophages. (A) RAW264.7 cells were stimulated with the indicated TLR stimuli or left untreated. After 3 hours, ATP release was measured. The means ± standard error of the mean (SEM) of 3 independent experiments are shown and statistical analysis is relative to naïve samples. (B) RAW264.7 cells were stimulated with LPS in absence or presence of 200 μg/mL 2-deoxyglucose and levels of ATP released were measured 3 hours later. The means ± standard deviation (SD) of triplicate determinations are shown and are representative of 2 independent experiments. (C) RAW264.7 cells left untreated or stimulated with LPS alone or in the presence of 50 μM ¹⁰Panx, 10 μM carbenoxolone, or 10 μM FFA. After 3 hours, ATP release was measured. The means of 3 independent experiments ± SEM are shown. (D) Levels of ATP released from LPS-stimulated RAW264.7 cells (open squares; dashed line) or BMDMs (closed squares; solid line) were measured over time. The means ± SEM of 3 independent experiments are shown. (E) The mRNA expression of Panx1 and Cd39 in RAW264.7 cells and BMDMs is shown. Data are representative of 3 independent experiments. (F) The surface expression of CD39 on RAW264.7 cells and BMDMs was determined by FACS and expressed as ΔMFI, compared with the isotype control for each group. The means ± SD of triplicate determinations are shown and are representative of 3 independent experiments. (G) CD39 expressed on peritoneal macrophages and BMDMs differentiated in the presence of L929 conditioned media, M-CSF, or GM-CSF for 7 days was determined by flow cytometry and expressed as ΔMFI, compared with isotype control for each group. The mean ± SD of triplicate determinations is shown and is representative of 2 independent experiments. (H) Flow cytometry analysis of CD39 surface expression (black histograms) on human monocytes (left) and macrophages (right) compared with isotype controls (gray histograms). Data are representative of 3 independent experiments. (I) Levels of ATP released from LPS-stimulated Cd39^−/− (open squares; dashed line) and wild-type (closed squares; solid line) BMDMs were measured over time. The means ± SEM of 3 independent experiments are shown. (J) The levels of ATP produced by Cd39^−/− BMDMs stimulated with LPS in the absence (open squares; dashed line) or presence (closed circles; solid line) of 50 μM ¹⁰Panx was measured over time. The means of 3 independent experiments ± SEM are shown. MFI, mean fluorescence intensity; WT, wild-type.