Ique3 flow cytometer

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).

A pCAGGS plasmid encoding CHIKV VLP (capsid-E3-E2–6K-E1) was subjected to mutagenesis of all residues of CHIKV E2 to alanine except for alanine residues, which were mutated to serine (Genscript). Select residues identified by alanine-scanning were additionally mutated to arginine with arginine and lysine residues mutated to glutamate (Genscript). Plasmids were transfected in a 96-well format with Expifectamine 293 into Expi293 cells. 24 h post-transfection, cells were stained with pre-titrated sub-saturating concentrations of mAbs for 30 min at 4°C followed by secondary staining with anti-human IgG Alexa Flour 647 for 20 min at 4°C. Cells were resuspended in FACS buffer containing DAPI for exclusion of dead cells. Data were acquired on an iQue3 flow cytometer, and analyzed with ForeCyt software (Sartorius).