Cell culture plastics

HEK293T (CRL-3216), HeLa (CCL-2), HEPG2 (HB-8065) and primary fibroblast cells (CCL-186) were all obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) through LGC Standards (Molsheim, France). The HEK293T stoplight cell line was constructed as described previously [34 (link)]. HEK293T stoplight cells, HEK293T HDR stoplight cells, HEK293T and primary fibroblast cells were cultured in a high-glucose DMEM medium, and HeLa and HEPG2 cells were cultured in Eagle’s minimum essential medium. The culture medium was supplemented with 10% (v/v) FBS (Sigma Zwijndrecht, The Netherlands), and cells were cultured at 37 °C and 5% CO₂. Cell culture plastics, unless otherwise specified, were purchased from Greiner Bio-One (Alphen aan de Rijn, The Netherlands).

HEK293T stoplight cells and HEK293T cells with stable EGFP expression were cultured in low-glucose DMEM medium supplemented with 10% fetal bovine serum (FBS), at 37 °C and 5% CO₂. The cell lines were both graciously gifted by Dr. Olivier de Jong and constructed as described previously, using the lentiviral plasmids containing the gene of interest (Stoplight construct [29 (link)] or EGFP [30 (link)]) in a pHAGE2-EF1a-IRES-PuroR or pHAGE2-EF1a-IRES-NeoR backbone, respectively. Alongside these lentiviral plasmids, HEK293T cells were transfected with pMD2.G plasmid, and PSPAX2 plasmid (Addgene #12259 and #12260, respectively) at a 2:1:1 ratio for lentiviral production. Lentiviral supernatant was then used to transduce HEK293T cells. To prevent multiple integrations of the fluorescent reporter constructs, HEK293T cells were transduced using an MOI < 0.1 and subsequently cultured and expanded with their respective selection antibiotics. After 2 weeks, cells were sorted using a BD FACSAria III cell sorter (Becton Dickinson, Franklin Lakes, NJ, USA), after which they were further expanded in the presence of selection antibiotics.
For subculturing between experiments, 1 mg/mL Gibco^® Geneticin^® Selective Antibiotic (G418 sulfate, Fischer Scientific, Landsmeer, The Netherlands) was supplemented. Cell culture plastics were acquired from Greiner Bio-One (Alphen aan de Rijn, The Netherlands).

[N-methyl-³H] acetyl-l-carnitine hydrochloride ([³H]-AC; 85 Ci/mmol) was purchased from American Radiolabeled Chemicals Limited (Herts, UK). Chemicals and all cell culture media and supplements were obtained from Sigma-Aldrich (Dublin, Ireland, or St. Louis, MO, USA), with the exception of ergothioneine, which was bought from Santa Cruz Biotechnology (Heidelberg, Germany). All cell culture plastics were obtained from Greiner BioOne (Frickenhausen, Germany).