Journal: The Journal of Biological Chemistry

Article Title: Oligomer Size of the Serotonin 5-Hydroxytryptamine 2C (5-HT 2C ) Receptor Revealed by Fluorescence Correlation Spectroscopy with Photon Counting Histogram Analysis

doi: 10.1074/jbc.M112.350249

Figure Lengend Snippet: Molecular brightness of fluorescence-tagged 5-HT 2C receptors expressed on the plasma membrane of hippocampal neurons and HEK293 cells Molecular brightness values were calculated from the FCS data by dividing the average photon count rate (obtained from the fluorescence intensity trace) by the number of fluorescent molecules (derived from the autocorrelation function) and are expressed as CPSM. Molecular brightness determined by PCH was performed using the PCH module in the Zeiss Aim 4.2 software for a one-component model, and the resulting reduced χ 2 value is reported. Data represent the mean ± S.E. for the number of cells indicated ( n ) from two or three independent transfection experiments.

Article Snippet: Histograms were constructed (using the PCH module in the Zeiss Aim 4.2 software) in which the number of 10-μs bins was plotted on the y axis, and photon counts were plotted on the x axis.

Techniques: Fluorescence, Clinical Proteomics, Membrane, Derivative Assay, Software, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Oligomer Size of the Serotonin 5-Hydroxytryptamine 2C (5-HT 2C ) Receptor Revealed by Fluorescence Correlation Spectroscopy with Photon Counting Histogram Analysis

doi: 10.1074/jbc.M112.350249

Figure Lengend Snippet: Photon counting histograms from FCS recordings made in the cytosol of HEK293 cells expressing a tandem, GFP-GFP dimeric construct (A) and the plasma membrane of HEK293 cells expressing 5-HT2C/GFP receptors (B). The inset shows the PCH on a linear scale. To generate a histogram, each 10-s fluorescence intensity trace (as illustrated in Fig. 3A) was broken down into 1 million 10-μs intervals or bins (PCH bin time = 10 μs). Histograms were constructed (using the PCH module in the Zeiss Aim 4.2 software) in which the number of 10-μs bins was plotted on the y axis, and photon counts were plotted on the x axis. The resulting histogram depicts the number of bins that registered 1,2,3 … n photon counts during one 10-s observation period. The histograms show the average number of photon counts per 10-μs bin time to be 1.24, equivalent to 124,000 counts/s. Dividing by the average number of molecules in the observation volume (7) yields an average molecular brightness of 17,714 CPSM. The residuals of the curve fit (shown in the lower panels) plot the number of bins on the y axis and photon counts on the x axis. The residuals show the deviation of the fit of the data to the selected model, providing a measure of how well the data fit the model. In this case, the data were fit to a one-component model for a single homogenous population of fluorescence-tagged receptors (i.e. dimers). The residuals of the curve fit are less than 2 S.D. values and are randomly distributed about 0, indicating that the data are a good fit for the selected model, with reduced χ2 equal to unity.

Article Snippet: Histograms were constructed (using the PCH module in the Zeiss Aim 4.2 software) in which the number of 10-μs bins was plotted on the y axis, and photon counts were plotted on the x axis.

Techniques: Expressing, Construct, Clinical Proteomics, Membrane, Fluorescence, Software

Journal:

Article Title: Clathrin-dependent pathways and the cytoskeleton network are involved in ceramide endocytosis by a parasitic protozoan, Giardia lamblia

doi: 10.1016/j.ijpara.2006.09.008

Figure Lengend Snippet: Quantitative analysis of confocal images. Fluorescence intensities for Bodipy-ceramide in the presence and absence of anti-cytoskeleton agent were measured by Zeiss LSM 5 PASCAL software version 3.2. Labeled trophozoites were randomly selected and analyzed as described Materials and methods. Results (fluorescence/area × 104) show that vinorelbine reduces ceramide uptake significantly.

Article Snippet: Histograms of fluorescence images (fluorescence/area × 10 4 , measured by Zeiss LSM5 PASCAL software version 3.2) in (C) show the relative amount of Bodipy-ceramide internalized by parasites in control and K + -depleted cells.

Techniques: Fluorescence, Software, Labeling

Journal:

Article Title: Clathrin-dependent pathways and the cytoskeleton network are involved in ceramide endocytosis by a parasitic protozoan, Giardia lamblia

doi: 10.1016/j.ijpara.2006.09.008

Figure Lengend Snippet: Potassium-depletion affects the colocalization of Bodipy-ceramide with anti-clathrin antibody. Both control and K+-depleted cells were first treated with Bodipy-ceramide (200 nM) for 30 min before reacting with anti-clathrin antibody. A) a) identifies clathrin-positive structures in trophozoites cultured in normal growth medium. b) is the 3D reconstruction of the same picture (a) using VIS-ART software (Carl Zeiss). Insets (a and b) show the association of vesicles with plasma membranes. B) c) demonstrates the localization of clathrin-positive vesicles in the presence of ceramide, which reveals enlargement of vesicles and migration towards endoplasmic reticulum/perinuclear regions. d) depicts Bodipy-ceramide labeling of trophozoites; and e) is the colocalization of ceramide with clathrin-coated vesicles in the endoplasmic reticulum/perinuclear regions. f, g) show clathrin-positive structures and Bodipy-ceramide labeling of K+-depleted cells. K+-depletion causes puffiness of trophozoites, illuminates discrete and isolated clathrin-like vesicles (f), and lowers ceramide uptake significantly (g). h) is the merger of images of (f) and (g). Histograms of fluorescence images (fluorescence/area × 104, measured by Zeiss LSM5 PASCAL software version 3.2) in (C) show the relative amount of Bodipy-ceramide internalized by parasites in control and K+-depleted cells. Arrowheads denote endoplasmic reticulum/perinuclear membranes and arrows indicate vesicle-like structures. Bars: 5 μM.

Article Snippet: Histograms of fluorescence images (fluorescence/area × 10 4 , measured by Zeiss LSM5 PASCAL software version 3.2) in (C) show the relative amount of Bodipy-ceramide internalized by parasites in control and K + -depleted cells.

Techniques: Control, Cell Culture, Software, Clinical Proteomics, Migration, Labeling, Isolation, Fluorescence