Cytoperm cytowash

Vero cells were infected with DENV1 TH-Sman (ATCC VR-1586), DENV2 New Guinea C (ATCC VR-1584), DENV3 H87 (BEI Resources) or DENV4 H241 (BEI Resources) at an MOI of 0.01. After 5 days, cells were harvested, then were fixed, permeabilized, and washed with Cytofix/Cytoperm and Cytoperm/Cytowash (BD Biosciences). Cells were incubated with sample containing human anti-DENV antibodies diluted in Cytoperm/Cytowash for 1 hr on ice, washed, and then stained with goat anti-human IgG Fc FITC (Abcam) for 1 hr on ice. After washing, cells were analyzed on a LSRII (BD Biosciences).

Vero cells were seeded in 100 μL of Medium 199 supplemented with 5% FBS and 1% PenStrep (Invitrogen) and plated at 5 × 10³ cells/well in flat-bottom 96-well plates. The next day, different dilutions of mAb-containing sample were incubated with 200 pfu/well of DENV for 1 hr at 37 ^oC. The neutralization mixture was then added to Vero cells. After 3 days, cells were harvested, then were fixed, permeabilized, and washed with Cytofix/Cytoperm and Cytoperm/Cytowash (BD Biosciences). Cells were stained with 4G2 diluted in Cytoperm/Cytowash for 1 hr on ice, washed, and then stained with goat anti-mouse IgG Fc FITC (Abcam) for 1 hr on ice. After washing, cells were analyzed on a LSRII (BD Biosciences). To assess antibody-dependent enhancement, K562 cells were seeded in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% FBS at 5 × 10³ cells/well in flat-bottom 96-well plates and incubated with neutralization mixture formulated as above. After 3 days, cells were fixed, permeabilized, and washed as above. Cells were stained with 4G2, washed, and then stained with goat anti-mouse IgG Fc FITC before a final series of washes. Cells were then analyzed on a LSRII as above.