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Results: 5

1.
Figure 4

Figure 4. From: Osmotic induction of calcium accumulation in human embryonic kidney cells detected with a high sensitivity FRET calcium sensor.

Calcium levels in HEK293T cells expressing GLUT1 or GLUT2 determined by TN-XXL. Bars indicate the loading time for external sugars (sucrose and glucose; 5, 25, and 40 mM, mannitol 40 mM) during perfusion with glucose-free Hanks’ balanced buffer in the presence of GLUT1 (A) or GLUT2 (B). FRET images were acquired and data were analyzed as described for Fig. 1. Data are shown as mean ± SD (n = 7-8).

Bi-Huei Hou, et al. Cell Calcium. ;46(2):130-135.
2.
Figure 2

Figure 2. From: Osmotic induction of calcium accumulation in human embryonic kidney cells detected with a high sensitivity FRET calcium sensor.

Sugar-induced calcium accumulation in HEK293T cells. Red (sucrose) and blue (glucose) bars indicate the loading time for external sugars (5, 25, and 40 mM) during continuous perfusion with glucose-free Hanks’ balanced buffer (A). FRET images were acquired and data were analyzed as described in Fig. 1. Data are shown as mean ± SD (n = 8). Bleed-through corrected ratio images are shown in pseudo-color ranging from blue (low) to red (high) (B). Bar corresponds to 10 μm.

Bi-Huei Hou, et al. Cell Calcium. ;46(2):130-135.
3.
Figure 3

Figure 3. From: Osmotic induction of calcium accumulation in human embryonic kidney cells detected with a high sensitivity FRET calcium sensor.

Cytosolic glucose levels in HEK293T cells using FLII12Pglu-700μδ6. Cells were perfused with different external glucose concentrations. Blue bars indicate the exposure time for a given bolus of external glucose (5, 25, and 40 mM) during perfusion with glucose-free Hanks’ balanced buffer in the absence (A) and in the presence of GLUT2 (B). FRET images were acquired and data were analyzed as described for Fig. 1. Data are shown as mean ± SD (n = 18-26). The bleed-through corrected ratio images are shown in pseudo-color ranging from blue (low) to red (high) (C). Bar is 10 μm.

Bi-Huei Hou, et al. Cell Calcium. ;46(2):130-135.
4.
Figure 5

Figure 5. From: Osmotic induction of calcium accumulation in human embryonic kidney cells detected with a high sensitivity FRET calcium sensor.

Sugar-induced volume change measurement in HEK293T cells expressing GLUT2 and analysis of calcium accumulation in HEK293T cells at physiologically relevant base glucose levels. (A) HEK293T cells expressing TN-XXL were perfused with different glucoses concentrations (data from Figure 2). Four images corresponding to four time points from each bolus were analyzed. The outline of the cells was determined from black and white images and the cell area was determined by pixel counting using ImageJ. (B) HEK293T cells expressing TN-XXL were perfused with a base glucose concentration of 4 mM and glucose boluses of 10, 25, and 40 mM were applied. Blue (glucose) bars indicate the loading time of external sugar during continuous perfusion with 4 mM glucose in Hanks’ balanced buffer.

Bi-Huei Hou, et al. Cell Calcium. ;46(2):130-135.
5.
Figure 1

Figure 1. From: Osmotic induction of calcium accumulation in human embryonic kidney cells detected with a high sensitivity FRET calcium sensor.

FRET analysis of intracellular calcium level with TN-XXL in HEK293T cells. Cells were perfused with 40 mM glucose (A-F). Open bars with gray columns indicate the bolus of external glucose during perfusion with glucose-free Hanks’ balanced buffer. Quantitative data were derived by pixel-by-pixel integration of the ratiometric images. The fluorescence intensity in arbitrary units (A.U.) for individual eCFP (ET470/24m) and Citrine (ET535/30m) emission channels were monitored with eCFP excitation (ET430/24x) and the FRET index for Venus and eCYP was determined (Y-axis corresponds to sensitized fluorescence Fc (background and bleed-through corrected using Citrine excitation (ET500/20x)) normalized to donor emission; note that peak intensities were used and that only the short wavelength emission peak of eCFP was considered for donor emission). FRET images were acquired every 5 sec and cytosolic calcium levels were analyzed. HEK293T cells were preincubated with 50 μM gadolinium (30 min) (B), 10 μM nifedipine (30 min) (C), 1 μM thapsigargin (15 min; D, 30 min; E) or no calcium (30 min) (F) without glucose. When steady-state calcium values were reached, a bolus of 40 mM glucose was added for 2 min. Data are shown as mean ± SD (n = 5-12).

Bi-Huei Hou, et al. Cell Calcium. ;46(2):130-135.

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