Alexa fluor 647 conjugated goat anti human or anti mouse igg

Vero cells were inoculated (multiplicity of infection [MOI] of 5) with alphaviruses in DMEM supplemented with 2% FBS. After allowing infection to proceed for 14 to 18 h, cells were detached using TrypLE (Thermo Fisher), washed with DMEM containing 2% FBS, and filtered through 100 μm nylon mesh (Corning). Cells then were incubated with 10 μg/mL of mAbs for 30 min at 4°C in FACS buffer. Cells were washed and incubated with Alexa Fluor 647 conjugated goat anti-human or anti-mouse IgG (1:2,000 dilution; Thermo Fisher) for 30 min at 4°C. Cells were washed, fixed with 2% paraformaldehyde (PFA), and subjected to flow cytometry analysis using an iQue3 flow cytometer (Sartorius).

Vero cells were inoculated (multiplicity of infection [MOI] of 1) with different alphaviruses in DMEM supplemented with 2% fetal bovine serum (Hyclone), 100 U/ml penicillin, 100 μg/ml streptomycin, and 10 mM HEPES. After allowing infection to proceed for 14 hours, cells were detached using TrypLE (Thermo Fisher) and washed with PBS. Cells then were incubated with serial dilutions of anti-alphavirus mAbs for 30 min at 4°C. Cells were washed and incubated with Alexa Fluor 647 conjugated goat anti-human or anti-mouse IgG (1:2000 dilution; Thermo Fisher) for 30 min at 4°C. Cells were washed, fixed with 2% paraformaldehyde (PFA), and subjected to flow cytometry analysis using a MACSQuant Analyzer (Miltenyi Biotec).