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2 protocols using vero e6 cells

1

DENV Viral Neutralization and ADE Assay

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Vero E6 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and were used for viral neutralization assays and to propagate DENV. Vero E6 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Welgene, Gyeongsan, Korea) supplemented with 5% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Grand Island, NY, USA). In addition, U937-DC-SIGN cells (ATCC, U937 cells transfected with the human DC-SIGN gene, encoding an attachment factor for DENV) were used for the DENV binding and ADE experiments. U937-DC-SIGN cells were cultured in Roswell Park Memorial Institute (RPMI) medium (Welgene) containing 5% FBS, 2 mM L-glutamine, 0.1 mM non-essential amino acids, and 0.05 mM 2-mercaptoethanol.
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2

Macrophage Polarization and Immune Cell Culture

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Human monocytic leukemic cell line THP-1 (Korean Collection for Type Cultures, KCTC; Jeongeup-si, Jeollabuk-do, Korea) was cultured in RPMI1640 medium (Welgene, Republic of Korea) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 1% penicillin/streptomycin (P/S), and 0.5 nM 2-mecaptoethanol. THP-1 monocytes were differentiated into Mɸs by treating with 25 nM PMA for 48 h prior to polarization. Human CD14+ monocytes (PromoCell, Heidelberg, Germany) were cultured with 50 ng/mL of GM-CSF or M-CSF for seven days to achieve differentiation. All Mɸs were polarized in vitro with either LPS (20 pg/mL; Invitrogen, USA) and IFN-γ (20 ng/mL) or IL-4 (20 ng/mL) and IL-13 (20 ng/mL) (Peprotech, USA) for 24 h. Cells were washed thrice with phosphate-buffered saline (PBS) to remove any remaining cytokines. Human PBMCs were purchased from Cellular Technology Limited (CTL; OH, USA). Vero E6 cells were purchased from KCTC and maintained in minimal essential medium (MEM; Welgene, Daejeon, Korea) supplemented with 10% FBS and 1% P/S.
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