Quantification of MDV Reactivation in CEF

The MDV reactivation was quantified by co-culturing pp38/pp24 activated 4523T with CEF as described previously with minor modification [26 (link)]. Briefly, 6 × 10⁵ of CEF cells per well were seeded into a 24-well plate on the day before co-culturing. Then, 10,000 gRNA transfected C4 cells with and without doxycycline treatment were co-cultivated with CEF for 24 h. After removing the C4 cells, the CEF cells were incubated for a further 5 days or until plaques had formed. The CEF monolayers were fixed with acetone/methanol for 5 min. The plaques were labelled with anti-gB mAb HB-3 [27 (link)] followed by the secondary antibody Rabbit Anti-Mouse Immunoglobulins/HRP (Agilent, Santa Clara, CA, USA). The plaques were developed using 0.1 M sodium acetate buffer pH 4.8, 3-amino-9-ethylcarbazole substrate solution (AEC, 4 mg/mL) and 30% hydrogen peroxide (Sigma-Aldrich, Burlington, MA, USA). The stained plaques were captured using an EVOS digital microscope (Thermo Fisher Scientific, Waltham, MA, USA) at 20× magnification.