Technical Reference #1906

1906.

Directional Spread of Surface-Associated Retroviruses Regulated by Differential Virus-Cell Interactions Nathan M. Sherer; Jing Jin; and Walther Mothes, King's College School of Medicine, Journal of Virology, 84(1906), (2010)
Link To Paper

Materials & Methods:
Live imaging and cellular manipulation. Live-cell imaging was performed at 37°C by using a 60 oil objective (numerical aperture 1.4) of a Nikon TE2000 inverted wide-field microscope equipped with Piezo drive or by using an Improvision spinning disc confocal microscope equipped with a Nikon TE2000 base as previously described (14 30). Cells were plated on 35-mm imaging dishes (MatTek Ashland MA) coated with poly-L-lysine or fibronectin in MEM with 10% FBS plus penicillin-streptomycin-glutamine as previously described (30). Timelapse acquisition of CFP or YFP channels was every 4 to 60 s as indicated in the legends to the supplemental movies. Fibronectin cytochalasin D nocodazole sodium azide Polybrene and heparinase II were obtained from Sigma-Aldrich (St. Louis MO). Drugs were administered at the concentrations indicated in the figure legends by 10-fold dilution into the culture medium during live-cell imaging. Exogenously administered fluorescent viruses (see Fig. 6 and 7) were generated as previously described (18) diluted in MEM containing 10% FBS and added directly to the culture medium. Coculturing of producer and target cells to monitor viral spread (see Fig. 7) was as previously described (14 30). All imaging experiments were carried out a minimum of three times in order to confirm reproducibility. Endogenous Gag fluorescence was pseudo-colored green and additional markers or exogenously administered viruses were pseudocolored red. All movies were edited using Openlab (PerkinElmer) and ImageJ (NIH) software packages and saved for presentation in Quicktime format using Sorensen 3 compression. Single-particle tracking was performed using Openlab or Volocity software packages (PerkinElmer). Particle assembly was monitored as previously described (14). The first peak of fluorescence achieving 85% of maximum intensity was chosen as a threshold representing the completion of particle assembly (defined as time [T] zero). Relative position and velocity were calculated for particle movements to or from the cell body as previously described (30).

Microscopic Technique
Inverted Wide-Field Microscopy

Cell Type(s)
Rat XC sarcoma cells; HEK293 cells