Genome-Wide Gene Expression Analysis Identifies the Proto-oncogene Tyrosine-Protein Kinase Src as a Crucial Virulence Determinant of Infectious Laryngotracheitis Virus in Chicken Cells

Li, Hai; Wang, Fengjie; Han, Zongxi; Gao, Qi; Li, Huixin; Shao, Yuhao; Sun, Nana; Liu, Shengwang

Flow cytometry and immunofluorescence. We conducted fluorescence-activated cell sorting (FACS) analyses using a BD FACScan flow cytometer and CellQuest software (version 4.0.2; BD, Mountain View, CA, USA). Cell death was assayed by examining cells in the sub-G 1 phase of the cell cycle via propidium iodide (PI) staining of permeabilized cells as previously described (41) . Apoptosis and necrosis were assayed with annexin V-PI using an annexin V-PI staining kit (Biyuntian, China) according to the manufacturer's instructions. Immunofluorescent detection of ILTV-positive cells was conducted on cells that had been fixed with 4% paraformaldehyde overnight. Fixed cells were incubated with a rabbit polyclonal antibody against ILTV glycoprotein I (gI), followed by a secondary goat anti-rabbit antibody conjugated to fluorescein isothiocyanate (FITC) (The Jackson Laboratory, Bar Harbor, ME, USA). A polyclonal antibody against the gI of ILTV was produced in a rabbit. Briefly, the rabbit was first immunized with the pCAGGS-gI vector via intramuscular injection, followed by boosting with inactivated ILTV and recombinant gI protein via intramuscular injection. The background level of reactivity was determined using control serum from nonimmunized SPF rabbits.