Pe cy 7 mouse anti human cd11c

PBMCs were stained with fluorescent dye conjugated to antibodies. For pDCs, BD Horizon Fixable Viability Stain 780 (FVS780) viability dye, PE-Cy7 Mouse Anti-Human CD123 (BD, 560826), BB515 Mouse Anti-Human Neuropilin-1 (CD304) (BD, 566036), APC Mouse Anti-Human CD86 (BD, 555660), and PE Mouse Anti-Human HLA-DR (BD, 556644) were used. For mDCs, FVS780, Anti-human Lineage Cocktail 1 (Lin1) (CD3, CD14, CD16, CD19, CD20, CD56) (BD, 340546), PE-Cy™7 Mouse Anti-Human CD11c (BD, 561356), APC Mouse Anti-Human CD86 (BD, 555660), and PE Mouse Anti-Human HLA-DR (BD, 556644) were used. After staining, both cell lines were fixed with BD Cytofix Fixation Buffer (BD Bioscience). CD123⁺CD304⁺ cells were defined as pDCs, and Lin1⁻ CD11c⁺ cells were identified as mDCs. The expression levels of HLA-DR and CD86 were used as activation markers of pDCs and mDCs. After staining, the cells were analyzed by flow cytometry using CytoFLEX (Beckman Coulter, Brea, CA, USA), and the data were analyzed using FlowJo 10.9.0 software (Treestar, ON, USA).

C20 cells (0.4 × 10⁶) were seeded in T25 tissue culture flask and incubated overnight at 37°C with 5% CO₂ and then infected with CHIKV at MOI 1. CHIKV was allowed to adhere for 1 h at 37°C with 5% CO₂. The unadsorbed virus was removed and the cells were washed once with fresh DMEM, and the cells were supplemented with complete medium – C20. Upon 24 hpi, the cells were trypsinized, pelleted and washed with PBS. Cells were stained in PBS using V500 Mouse Anti-Human HLA-DR (Cat-561224; BD Biosciences), V450 Mouse Anti-Human CD14 (Cat-560349; BD Biosciences) and PeCy7 Mouse Anti-Human CD11c (Cat-561356; BD Biosciences) antibodies for 20 minutes as per manufacturer’s instructions. FACSVerse flow cytometer (BD Biosciences) was used to acquire the data followed by data analysis on FlowJo (BD Biosciences) software. The experiments were performed in triplicates.