Hek 293t kidney cells

Huh-7.5 cells (kind gift from Charles Rice), HEK-293T kidney cells (ATCC CRL-3216), TE-671 cells (ATCC CRL8805), A549 cells (kind gift from P. Boulanger), VeroE6 cells (ATCC CRL-1586), EBL cells (kind gift from Fabienne Archer), MDBK cells (European Collection of Authenticated Cell Cultures (ECACC)) were grown Dulbecco’s modified minimal essential medium (DMEM, Invitrogen, France) supplemented with 100 U/mL of penicillin, 100 µg/mL of streptomycin and 10%. of fetal bovine serum.
PHH (BD Biosciences) were centrifuged in F12-HAM medium (Sigma Aldrich) and seeded overnight in collagen-coated plates in BD Gentest seeding medium supplemented with 5% FCS. 16 h later, PHH were washed and cultured with a culture medium for PHH (DMEM F12, Sigma Aldrich) supplemented with 10% FCS, 1 µg/mL BSA, 5 µg/mL bovine insulin, 1 × 10⁻⁶ M Dexamethasone (Sigma Aldrich), 1 × 10⁻⁸ M 3.3 trilodo-L-thyronin, 5 µg/mL apotransferrin, 1% of non-essential amino acids (Gibco), 1% of Glutamin (Gibco) and 1% Penicillin-Streptomycin solution (Gibco).

The assay was performed as described previously [36] (link). HEK293T kidney cells (ATCC CRL-1573) were cultured in DMEM supplemented with 10% FBS, 1% non-essential amino acids, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells (2.5×10⁵ cells/well seeded in 35 mm 6-well tissue culture dishes 24 h before transfection) were co-transfected using the calcium phosphate reagent with a plasmid encoding HCV envelope proteins (H77 strain genotype 1a) or Chikungunya envelope encoding-plasmid (ChikV) issued from ChikV E3E1E2 plasmid from the Réunion infectious clone and with an HIV-1 LTR (long terminal repeat) luciferase reporter plasmid (kindly provided by Francoise Bex). After 12 h, transfected HEK293T cells were detached with 0.53 mM EDTA (Invitrogen) and co-cultured (5×10⁴ cells/well) with Huh-7-Tat indicator cells (5×10⁴ cells/well). Co-cultured cells were then incubated with 1 μM of GW3965 or with DMSO. After 24 h, the cells were washed with serum-free DMEM, incubated for 3 min in either pH 7 or pH 5 buffer, (containing 130 mM NaCl, 15 mM sodium citrate, 10 mM MES and 5 mM Hepes), then washed with serum-free DMEM. Cells were next incubated with 1 μM of GW3965 or with DMSO for 48 h. Luciferase activity was measured using a luciferase assay kit (Promega). The experiments were performed several times and results interpreted using student t-test.