Fitc conjugated cd86

DENV-2/16681 was added to 80% confluent C6/36 cells at an MOI of 0.01 in RPMI medium RPMI supplemented with 2% fetal calf serum, 10 U/ml penicillin, 10 mg/ml streptomycin (all Invitrogen) and 2 mM L-glutamine (Lonza). After 5–7 days, supernatant was harvested and cleared from cellular debris by centrifugation and subsequent filtration using a 0.2 μM filter. Supernatant was aliquoted, snap-frozen in liquid nitrogen and stored at -80°C. Viral titers were determined as described previously[57 ].
DCs were infected with DENV at an MOI of 1 unless stated otherwise. Infection was determined after 36-48h by flow cytometry. Cells were fixed in 4% para-formaldehyde for 15 min followed by permeabilization in PBS supplemented with 0.1% saponin for 10 min. Cells were stained with anti-NS3 (1:800, SAB2700181, Sigma) followed by PE-conjugated anti-rabbit (1:200; 711-116-152, Jackson ImmunoResearch) in combination with APC-conjugated CD83 (1:25, 551073, BD Pharmingen) and FITC-conjugated CD86 (1:25, 555657, BD Pharmingen). Cells were analyzed on a FACS Canto II (BD Biosciences).

Human monocytes isolated with CD14 magnetic beads (Miltenyi Biotec) from human PBMCs were exposed for 5 days in the presence of ACM. Cells were then collected and stained with APC-conjugated CCR2 (Biolegend, diluted 1:50), PE-conjugated CD14 (Biolegend, diluted 1:100), APC-conjugated HLADR (Biolegend, diluted 1:50), FITC-conjugated CD80 (BD pharmingen, diluted 1:50) and FITC-conjugated CD86 (BD pharmingen, diluted 1:50) after the FcR block for 15 min (Miltenyl Biotec, diluted 1:100). In high CD14 expressing cells, we examined the intensity with the other antibody. The mean fluorescence intensity (MFI) of each sample was analyzed with Canto II flow cytometer (BD biosciences).

The purity and phenotype of modified-DC was analysed using FACS Canto II flow cytometer (BD Biosciences, USA). Cells were stained with fluorescein isothiocyanate(FITC)-conjugated CD86, phycoerythrin(PE)-conjugated CD80, CD14, and HLA-DR, allophycocyanin(APC)-conjugated CD83, CD40, CD54, and HLA-ABC monoclonal antibodies (BD Biosciences, USA), and PE-conjugated CCR7 monoclonal antibody (R&D systems, USA). FITC-, APC-, PE- mouse isotype immunoglobulins, and PE mouse anti-human HLA-DR were used as background controls.