C6/36 Aedes albopictus cells were inoculated with a MOI 0.01 of ZIKV (H/PF/2013) or different DENV serotypes (Nicaraguan strains DENV-1 1254-4, DENV-2 172-08, DENV-3 N2845-09, DENV-4 N703-99). At 120 h post infection, cells were fixed with 4% PFA diluted in PBS for 20 min at room temperature and permeabilized with HBSS supplemented with 10 mM HEPES, 0.1% saponin and 0.025% NaN3 for 10 min at room temperature. 50,000 cells were transferred to U-bottom plates and incubated for 30 min at 4 °C with 5 μg ml−1 of anti-ZIKV human mAbs or negative (hCHK-152)12 (link), or positive (hE60)30 (link) isotype controls. After washing, cells were incubated with Alexa-Fluor-647-conjugated goat anti-human IgG (Invitrogen) at 1:500, fixed in 1% PFA in PBS, processed on MACSQuant Analyzed (Miltenyi Biotec), and analysed using FlowJo software (Tree Star).
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ZIKV and DENV Antibody Binding in Aedes albopictus Cells
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Corresponding Organization : Vanderbilt University
Other organizations : Vanderbilt University Medical Center, Washington University in St. Louis, Integral Molecular (United States)
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Variable analysis
independent variables
- MOI (multiplicity of infection) of ZIKV (H/PF/2013) or different DENV serotypes (Nicaraguan strains DENV-1 1254-4, DENV-2 172-08, DENV-3 N2845-09, DENV-4 N703-99)
- ZIKV or DENV infection of C6/36 Aedes albopictus cells
- Cell line: C6/36 Aedes albopictus cells
- Fixation and permeabilization conditions: 4% PFA diluted in PBS for 20 min at room temperature, HBSS supplemented with 10 mM HEPES, 0.1% saponin and 0.025% NaN3 for 10 min at room temperature
- Incubation conditions: 50,000 cells transferred to U-bottom plates and incubated for 30 min at 4 °C
- HE60 - anti-Dengue virus mAb
- HCHK-152 - negative isotype control
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