nonlinear regression plot  (GraphPad Software Inc)

Journal: International Journal of Molecular Sciences

Article Title: Examination of Intracellular GPCR-Mediated Signaling with High Temporal Resolution

doi: 10.3390/ijms23158516

Figure Lengend Snippet: Stopped-flow experiments with DmOctα1B- and DmOctα1B-GCaMP3.0-expressing cell lines. Fluo-4-loaded DmOctα1B ( A ) and DmOctα1B-GCaMP3.0 ( B ) cells were stimulated with increasing octopamine concentrations ranging from 10 −10 –10 −6 M in stopped-flow measurements. Fluorescence was monitored over 40 s and fluorescence changes (ΔF/F 0 ) for each octopamine concentration were calculated and plotted against the time ( A1 , B1 ). To resolve the initial signal response times, the first 4 s of each measurement were analyzed and are shown ( A2 , B2 ). Representative measurements from three independent datasets are shown. Each data point was obtained from triplicate measurements; ( C ) Concentration–response curves were established by plotting maximal changes in fluorescence against octopamine concentrations. Maximal changes in fluorescence ((F max − F 0 )/F 0 = ΔF/F 0 ) at the highest octopamine concentration were normalized to 100% and EC 50 values were obtained from nonlinear fitting of the data using GraphPad Prism v5.04. A representative concentration–response curve is shown. Mean EC 50 values are indicated from three independent datasets; ( D ) Bar graph indicating the time (s) passed until fluorescence signals were detected with Fluo-4-loaded (green) and GCaMP3.0-expressing (grey) cells (y-axis) and plotted against octopamine concentrations. Mean values ± SEM from three independent datasets are shown.

Article Snippet: The EC 50 values were determined from a nonlinear regression plot (four parameters) using GraphPad Prism v5.04 for analysis and display.

Techniques: Expressing, Fluorescence, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Examination of Intracellular GPCR-Mediated Signaling with High Temporal Resolution

doi: 10.3390/ijms23158516

Figure Lengend Snippet: Stopped-flow experiments with flpTM-DmOctβ1 cells loaded with Fluo-4. Fluo-4-loaded flpTM-DmOctβ1 cells were stimulated in stopped-flow experiments with increasing octopamine ( A ) and NKH477 ( B ) concentrations. Fluorescence intensities were monitored over 80 s and 100 s, respectively. Fluorescent changes (ΔF/F 0 ) for each octopamine and NKH477 concentration were calculated and plotted over time ( A1 , B1 ). To resolve signal response times, the initial 35 s and 40 s of each measurement are displayed ( A2 , B2 ). Shown are representative measurements from three independent datasets. Each data point was obtained from triplicate measurements; ( C ) Concentration–response curves were generated by plotting maximal changes in fluorescence against octopamine (green) and NKH477 (red) concentrations. Maximal changes in fluorescence at the highest ligand concentrations were normalized to 100% and EC 50 values were obtained from nonlinear fitting of the data using GraphPad Prism v5.04. Shown is a representative concentration–response curve from three independent datasets. Mean EC 50 values are indicated; ( D ) Bar graph showing the time until signal was detected at concentrations in the range of EC 50 values (dynamic range) and at saturating concentrations (saturating range) for octopamine (green) and for NKH477 (red). Each data point was obtained from triplicate measurements. Shown are mean values ± SEM from three independent datasets.

Article Snippet: The EC 50 values were determined from a nonlinear regression plot (four parameters) using GraphPad Prism v5.04 for analysis and display.

Techniques: Fluorescence, Concentration Assay, Generated

Journal: International Journal of Molecular Sciences

Article Title: Examination of Intracellular GPCR-Mediated Signaling with High Temporal Resolution

doi: 10.3390/ijms23158516

Figure Lengend Snippet: Stopped-flow experiments with FlpTM-DmOctβ1-GCaMP3.0 cell lines. FlpTM-DmOctβ1-GCaMP3.0 cells were stimulated with increasing octopamine ( A ) and NKH477 ( B ) concentrations. Fluorescence intensities were monitored over 100 s. Fluorescence changes (ΔF/F 0 ) for each octopamine and NKH477 concentration were calculated and plotted over time ( A1 , B2 ). To resolve signal response times, the initial 35 s and 40 s of each measurement are shown ( A2 , B2 ). Shown are representative measurements from three independent datasets. Each data point was obtained from triplicate measurements; ( C ) Concentration–response curves were generated by plotting maximal changes in fluorescence against octopamine (green) and NKH477 (red) concentrations. Maximal changes in fluorescence at the highest ligand concentrations were normalized to 100% and EC 50 values were obtained from nonlinear fitting of the data using GraphPad Prism v5.04. A representative concentration–response curve from three independent datasets is shown. Mean EC 50 values are indicated; ( D ) Bar graph showing the time until signal was detected at concentrations in the range of EC 50 values (dynamic range) and at saturating concentrations (saturating range) for octopamine (green) and for NKH477 (red). Each data point was obtained from triplicate measurements. Shown are mean values ± SEM from three independent datasets.

Article Snippet: The EC 50 values were determined from a nonlinear regression plot (four parameters) using GraphPad Prism v5.04 for analysis and display.

Techniques: Fluorescence, Concentration Assay, Generated