T rex hek293 cells

HEK293 T-Rex cells were purchased from Invitrogen. C5 cells contain a bidirectional artificial heat shock promoter with 8 times mulitmerized HSEs driving the expression of firefly luciferase and GFP and were previously described [27 (link)]. XshHSF1-5-13 HSF1 knock-down cells were generated by lentiviral transduction of HEK293 T-Rex cells with MISSION shRNA lentiviral particles (TRCN0000318712, Sigma Aldrich) and single clone selection. All cell lines were kept in an incubator at 37°C with 5% CO₂ in Dulbecco’s modified eagle medium (DMEM) with 4.5 g/L glucose, Na-Pyruvate L-glutamic acid, 10% fetal bovine serum and 1 x Penicillin / Streptomycin. DMEM containing all the before mentioned substances will be called DMEM complete hereafter. For experiments in 96-well plates, the plates were coated with polyethyleneimine (PEI) [31 (link)] before seeding. Transient transfections were conducted as described in [28 (link)]. For microscopy cells were seeded in a 6-well plate and treated with cadmium in the same way as for luciferase reporter assays. After 48 h cells were stained with PI (1 ng/μL) for 30 min before imaging with a Zeiss Observer microscope. P-values were calculated by the Student’s t-test. Statistical significance: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Conditional expression of CCR5 and ACKR2 was achieved using HEK293 T-Rex cells (Life Technologies), which stably express the tetracycline-responsive repressor protein and inhibit gene expression downstream to tetracycline-responsive operon. HEK293 T-Rex cells were maintained in complete D-MEM with 25 μg/ml blasticidin and were transfected using lipofection (Lipofectamine 2000, Invitrogen) with pcDNA4/Tet-on plasmids encoding HA-tagged ACKR2 and CCR5 under control of a tetracycline-responsive promoter. Cells were selected using 100 μg/ml zeocin (Life Technologies) and receptor expression was induced incubating cells with 1 μg/ml tetracycline. Analysis was performed 24 h after receptor expression induction.

T225 CellBind cell culture flasks (10×; Corning) were seeded with approximately 5 × 10⁶ human embryonic kidney 293 (HEK-293) T-REx cells (Invitrogen) each. Cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin (Gibco) until 80% confluent. Cells were infected with ChAdOx1.eGFP, provided by the Coughlan Lab (University of Maryland), at a multiplicity of infection of 0.01. Cells were then monitored for cytopathic effect (CPE). When culture medium turned yellow, medium was replaced. Once CPE became evident, but the cells were not ready to be collected, yellowed medium was supplemented with sodium bicarbonate buffer (pH 7.4, Gibco) until it reddened. This was done to retain the virus released into the medium. Once CPE was observed in >80% of the cell monolayer, the cells (5 to 8 days after infection) were dissociated from the flask by knocking. The supernatant and cells were separated by centrifugation at 300g for 5 min, both were stored at −80°C until ready for purification.