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Fig. 5 | Molecular Neurodegeneration

Fig. 5

From: Ex vivo imaging of active caspase 3 by a FRET-based molecular probe demonstrates the cellular dynamics and localization of the protease in cerebellar granule cells and its regulation by the apoptosis-inhibiting protein survivin

Fig. 5

FRET measurements of Casp3 activity in live OCCs. a-b Repetitive imaging with a 40x lens results in an artifact increase of the ECFPem/Venusem values recorded from the same cells in a 24-h interval. a progressive increase in the ECFPem/Venusem ratio in six CGCs monitored at subsequent intervals up to 24 h. b comparison of the ECFPem/Venusem ratios in two groups of cells undergoing single (left - blue dots) or repeated (right - red dots) laser excitation to capture FRET pair images. c Imaging with a 20x lens and repetitive laser excitations did not significantly alter the ECFPem/Venusem ratio in seven CGCs that were photographed up to ten times to acquire FRET pair images. d-f Exemplificative traces of the fluctuations in the ECFPem/Venusem ratio in healthy (d), suffering (e) or dying/dead (f) CGCs during a four-hour follow up. FRET was measured in these eleven cells during the course of the same experiment. Cell 2 and 3 in f died soon after one hour from start, and could not be subsequently identified when the culture was scanned for the acquisition of images at 120 m. It should be noted that live imaging can be made difficult by X-Y axis and focus (Z axis) drifting of the sample. If in-focus images cannot be captured at all time-points, cell(s) must be discharged from subsequent analysis. g = Effect of 60 mM KCl depolarization on the ECFPem/Venusem ratio during a three-hour follow up (see text for further explanation). h Exemplificative traces of the fluctuations in the ECFPem/Venusem ratio during a three-hour follow up in eight CGCs challenged with 100 mM H2O2. i Lack of a statistically significant response to 100 mM H2O2 in a subpopulation of CGCs that appeared to be resistant to oxidative stress (see text for discussion). Error bars = SEM. * P-value 0.05–0.01 %

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