Technical Reference #1855

1855.

Effect of ATP Binding and Hydrolysis on Dynamics of Canine Parvovirus NS1 Einari A. Niskanen; Teemu O. Ihalainen; Olli Kalliolinna; Milla M. Ha¨kkinen; and Maija Vihinen-Ranta, University of Jyväskylä, Journal of Virology, 84(1855), (2010)
Link To Paper

Materials & Methods:
Live-cell imaging and fluorescence recovery after photobleaching. In live-cell imaging experiments cells were cultivated on glass-bottom dishes (MatTek Ashland MA) and transfected 20 h before experiments. Confocal laser scanning microscopic images were acquired with a Zeiss LSM 510 inverted laser scanning microscope (Carl Zeiss AG Jena Germany) using a Plan-Neofluar 63 (NA 1.25) oil immersion objective. The sample holder and the objective were heated to 37°C. Optical sections from the middle of the nucleus were obtained with an image size of 256 by 256 pixels and a resolution of 100 nm/pixel. A 514-nm laser line and a 560- to 600-nm-band-pass filter were used to acquire the EYFP signal. In half-nucleus FRAP (hnFRAP) experiments seven successive iterations of high laser intensity (100% of 25 mV) were used to bleach approximately half of the nucleus. Images were acquired with low laser intensity (0.5 to 1%) and an appropriate time interval (0.5 to 2 frames per s [fps]). The optimal imaging rate was determined in preliminary studies and was 4 fps for K406M-deYFP and EYFP 1 fps for E444Q-deYFP and R510A-deYFP and 0.5 fps for NS1-deYFP R508A-deYFP and E445Q-deYFP. Image data were processed as described for fixed samples and were analyzed with ImageJ and spreadsheet software. FRAP data were normalized as follows (49): FREL (Bt/B0)/(Nt/N0). Relative fluorescence (FREL) was obtained from the fluorescence of the bleached area at time point t (Bt) the average fluorescence of the bleached area before bleaching (B0) total nuclear fluorescence at time point t (Nt) and average nuclear fluorescence before bleaching (N0). Background fluorescence was measured from outside the cell and subtracted from all values. Half-time recovery (t[1/2]) was the median value between the lowest and highest values after bleaching. Student’s t test was used to evaluate the significance of differences in half-time values.

Microscopic Technique
Confocal laser scanning microscopy

Cell Type(s)
NLFK cells