Human primary retinal pigmented epithelial (RPE) cells (Lonza, Walkersville, MD) were cultured in RtEGM™Bulletkit® media as per manufacturer’s instruction (Lonza). Vero cells (ATCC CCL-81) and Aedes albopictus clone C6/36 cells (ATCC CRL-1660) were cultured in DMEM, and EMEM media supplemented with 10% FBS, 10 µg/ml L-glutamine, and 1% penicillin and streptomycin solution as per manufacturer’s recommendation. ZIKV Virus (ZIKV) strain PRVABC59, NR-50240, originally isolated from human blood in Puerto Rico in December 2015, and DENV type 2 strain NR12216 were obtained through BEI Resources, NIAID, NIH. This ZIKV strain is 97–100% genetically similar to the current ZIKV strain circulating in Brazil57 (link). ZIKV and DENV were propagated in ATCC CCL-81 Vero cells and ATCC CRL-1660 C6/36 cells respectively, and titers were determined by plaque assay. RPE cells were infected with ZIKV and DENV at MOI of 1 for the indicated time points as described previously7 . For ABCG1 functional studies, Pr. RPE cells were treated with a pharmacological inhibitor of ABCG1, Benzamil (50 µM) (Tocris Biosciences, Minneapolis, MN), and cholesterol-water soluble (10 µM) (Sigma Aldrich, St Louis, MO) 1 h before ZIKV challenge.
Aedes albopictus clone c6 36 cells
Manufactured by American Type Culture Collection
Aedes albopictus clone C6/36 cells are an immortalized cell line derived from the Asian tiger mosquito, Aedes albopictus. These cells are commonly used in research applications involving arboviruses and other mosquito-borne pathogens.
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Lab products found in correlation
Water soluble cholesterol, by Merck Group (1 mentions)
Vero cell, by American Type Culture Collection (1 mentions)
Rtegm bulletkit, by Lonza (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by American Type Culture Collection (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
L glutamine, by American Type Culture Collection (1 mentions)
Tryptose phosphate, by Thermo Fisher Scientific (1 mentions)
5 protocols using aedes albopictus clone c6 36 cells
1
Zika and Dengue Virus Infection in RPE Cells
2
ZIKV Infection of hPSC-derived Neural Cells
3
ZIKV Infection of hPSC-derived Neural Cells
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The ZIKV PRVABC59 strain (ZIKVPR) was obtained from ATCC (Manassas, VA) and subsequently amplified in Aedes albopictus clone C6/36 cells (ATCC). Briefly, C6/36 cells were inoculated with viral inoculum for 1 h at 28 °C in a low volume of medium (3 mL per T-75 flask), with rocking every 15 min, before the addition of an additional 17 mL medium. Virus-inoculated cells were then incubated at 28 °C for 6–7 d before harvesting the supernatant. C6/36-amplified ZIKVPR titer was determined by infecting Vero cells for 48 h with a methylcellulose overlay and analyzed for focus-forming units per mL (FFU/mL). In mock infections, an equal volume of spent uninfected C6/36 culture medium was used. hPSC-derived hNCCs were seeded at a density of 50 cells/mm2 and maintained for 2–4 days prior to ZIKV infection. hNCC-derived hPNs were seeded at a density of 1700 cells/mm2 and matured for 2–3 weeks prior to ZIKV infection. Then these cells were infected with ZIKVPR at MOI of 0.04 or 0.4 and analyzed at 65 h post infection after three times washing the cells with culture media or PBS. The experimenter was not blinded to treatment. None of cell cultures were excluded from our analyses.
4
Cell Culture and Plaque Assay Protocol
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Human primary trabecular meshwork (TM) (69 (link), 70 (link)) cells and the human GTM3 cell line (69 (link)) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 µg/ml l -glutamine, and 1% penicillin-streptomycin (Thermo Scientific, Rockford, IL) solution. Vero cells (ATCC CCL-81) and Aedes albopictus clone C6/36 cells (ATCC CRL-1660) were cultured in DMEM and Eagle’s minimal essential medium (EMEM), respectively, supplemented with 10% FBS, 10 µg/ml l -glutamine, and 1% penicillin-streptomycin solution per the manufacturer’s recommendation. All the cells (except C6/36, maintained at 28°C) were maintained at 37°C with 5% CO2. The plaque assay was performed using Vero cells as described in one of our recent studies (25 (link)).
5
West Nile Virus Strain Production
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The WNV strain used in this work belongs to lineage 1 and was isolated from a human brain during the epidemic that occurred in Tunisia in 1997 (kindly gifted by the French National Reference Center on Arboviruses, Marseille, France). The viral stock was produced on the Aedes albopictus clone C6/36 cells (ATCC CRL-1660). Insect cells were cultivated in Leibovitz’s L-15 medium (Gibco, Waltham, MA, USA; Catalog No. 11415049) supplemented with 2% of tryptose phosphate (Gibco, Waltham, MA, USA; Catalog No. 260200) and 5% of FBS in 75-cm2 tissue culture flask at 28 °C until 50% of confluency and then infected at a multiplicity of infection (MOI) of 0.01 for 72 h. Cell supernatants were clarified by centrifugation for 15 min at 1500× g and frozen at −80 °C in cryotubes containing 500 μL of Leibovitz’s L-15 medium supplemented with 0.5 M sucrose and 50 mM HEPES. The final titer of the viral suspension was 107.97 TCID50 (median tissue culture infection dose) per mL as determined by plaque assays on Vero cell monolayers as described below.
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