TRPV1 Activates ROS Production - Live Cell Imaging. A-D Clonal SW982 Synoviocytes. A, B- Live cell ROS imaging in SW982 synoviocytes before (A) and 15 min after (B) activation of TRPV1 with resiniferatoxin (100 nM). At least five of the cells in this view (arrows) demonstrated a significant increase in DCF fluorescence over time, reflecting a TRPV1-mediated oxidative burst. C - Quantification of fluorescent emission for the ROS response to resiniferatoxin over time (min) is shown in the plot. Cells were pre-loaded with 4 μM 5-(and-6)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) for 20 min at room temperature. The cellular uptake and ROS converted 5-(and-6)-carboxy-2',7'-dichlorofluorescein (DCF) fluorescence was measured by live cell imaging for 30 min. Resiniferatoxin (100 nM) was added by bath perfusion for 4 min as indicated by the arrow. Graph shows typical fluorescence signal acquired from one responsive and one non-responsive cell out of 48 simultaneously recorded cells. D - Averaged DCF fluorescent emission responses to saline, capsaicin (1 μM), or capsaicin plus TRPV1 antagonist SB-366791 (30 μM) are shown in the plot (arbitrary units) over time (min). Fluorescent emission was significantly increased in cells activated with capsaicin. ROS induced fluorescent DCF emission in cells pre-treated for 10 min with the TRPV1 antagonist and then activated with capsaicin were not significantly increased over saline treated cells. Numbers of cells are indicated in parenthesis averaged from three experiments. E-F TRPV1 Mediated ROS Production in Primary Human Synoviocytes from Two Patients. Live cell imaging was used to visualize ROS generation over time (min) after stimulation with TRPV1 agonist resiniferatoxin and conversion of a ROS sensitive dye to fluorescent DCF in the synoviocytes. Primary human synoviocytes were acclimated 3 weeks in culture after harvesting from knee joints of patients with active arthropathies (osteoarthritis, pseudogout). Resiniferatoxin (100 nM) was added in bath perfusion for 4 min at the time indicated by arrow. Fluorescent image (E) and quantitative graph (F) show typical fluorescence signal acquired from one responsive (b) and one non-responsive (a) out of 85 simultaneously recorded cells. A total of 68 cells responded to the TRPV1 agonist out of 162 total cells imaged from the two patients.