Inverted florescence microscope

Cells were plated at 5 × 10³ per well in 96-well microtiter plates, treated with different cytokines and incubated for 24 hrs. Then 100 μl 50 μM 5-ethynyl-2′-deoxyuridine (EdU) (CellLight EdU DNA imaging Kit, Guangzhou RiboBio, China) were added into each well and the cells were cultured for an additional 2 hrs. The cells were stained with EdU according to the manufacturer's protocol. EdU medium was discarded and 4% paraformaldehyde was added to fix the cells at room temperature for 30 min. The cells were washed with glycine (2 mg/ml) for 5 min in a shaker and treated with 0.5% Trion X-100 for 10 min before washed with PBS for five minutes. The cells were then incubated with 1× Apollo® click stain reaction buffer for 30 min while protecting from light, washed with 0.5% Triton X-100 for three times to permeate the cells, stained with Hoechst33342 (10ug/ml) for 30 min at room temperature, washed with PBS for three times. The cells were examined with an inverted florescence microscope(Olympus, Japan) immediately.

Chinese Hamster Ovary (CHO) cells were transfected with pVAX1-GFP-ROP1 plasmid (3 μg/μl) using TurboFect (Fermentas Inc., Glen Burnie, MA, USA) according to the manufacturer’s instructions. Expression of green fluorescent protein (GFP)-tagged ROP1 was observed under inverted florescence microscope (Olympus, Hicksville, NY) at 24, 48, and 72 h post transfection. The expression was also detected by western blot assay using monoclonal anti-GFP antibody (Invitrogen, Grand Island, NY, USA) and toxoplasmosis-positive patient serum (Diagnostic Laboratory [Para: SEAD], Department of Parasitology, University of Malaya). CHO cells transfected with pVAX1-GFP vector was included as positive control.