Technical Reference #1872

1872.

Equine herpesvirus type 1 (EHV-1) utilizes microtubules; dynein; and ROCK1 to productively infect cells Arthur R. Frampton Jr.; Hiroaki Uchida; Jens von Einem; William F. Goins; Paola Grandi; Justus B. Cohen; Nikolaus Osterrieder; Joseph C. Glorioso, University of Pittsburgh School of Medicine, Veterinary Microbiology, 141(1872), (2010)

Keywords:
EHV-1; Trafficking; Microtubules; Dynein; ROCK1

Materials & Methods:
CHO-K1 cells were seeded to confluency in collagencoated 35mm dishes (MatTek Corporation Ashland MA). Cells were mock-treated or treated with 100 mM Y-27632 or 10mM paclitaxel for 30 min at 37 8C. Cells were chilled on ice for 10 min and then L11VP26mRed was added to the cells at an MOI of 10. Virus was incubated on the cells at 4 8C for 2 h in the presence or absence of drug. After 2 h cells for the 0 time-point were fixed with 4% paraformaldehyde (PFA) at 4 8C. Media pre-warmed to 37 8C with or without drug was added to the remaining cells and then the cells were fixed with 4% PFA at 15 30 60 and 120 min post-temperature shift. After 30 min of fixation with 4% PFA cells were rehydrated with PBS for 15 min at 4 8C. Cells were washed once more with PBS for 15 min and then cells were examined using an inverted Olympus Fluo-View 1000 confocal microscope (Olympus America Inc. Center Valley PA). Prior to imaging cells were stained with Hoechst dye (1mg/mL) and wheat germ agglutinin (5mg/ mL) to label the nucleus and plasma membrane respectively. All images were captured at a 60 magnification.

Microscopic Technique
Confocal Microscopy

Cell Type(s)
CHO-K1 cells