Lab tek chambered coverglass systems

HeLa Kyoto (RRID:CVCL_1922) and HeLa rCDS cells (24) were cultured at 37°C and 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% fetal calf serum (FCS; Biochrom), 100 U/mL penicillin, and 100 µg/mL streptomycin. The cell line identity of HeLa Kyoto cells has been authenticated using STR profiling (Promega PowerPlex 21 Kit; carried out by Eurofins Genomics, Ebersberg, Germany). Cell lines were grown from mycoplasma-free liquid nitrogen stocks. Passaged cells in culture in the lab are monitored regularly (every 4 months) for mycoplasma contamination using the MycoAlert mycoplasma detection kit (Lonza, Rockland, USA), and cell lines used here were contamination-free. For microscopy experiments, cells were seeded on slides within eight-well Lab-Tek chambered coverglass systems (Thermo Scientific, Waltham, USA) or Ibidi µ-Slide 8 Well Glass bottom slides (Ibidi, Planegg, Germany). At about 50% confluence, cells were transfected using Turbofect (Thermo Scientific, Waltham, USA) according to the manufacturer's instructions. 0.5 or 0.75 µg DNA was transfected per Labtek or Ibidi well, respectively. Tagged pCHIV derivatives were transfected in an equimolar ratio with their non-labeled counterpart. At 4 hpt, the transfection mixture was replaced by fresh medium.