Endocytosis Assay in Human Kidney Cells

Human kidney proximal tubular epithelial HK-2 cells originate from the American Tissue Type Collection (ATTC, USA) and were used to study the endocytosis previously [25 (link)]. Human conditionally immortalized PTEC (ciPTEC) were obtained from living kidney cells exfoliated into urine as described previously [26 (link)]. Proximal tubular cells were grown in DMEM-HAM’s F12 culture medium (Lonza) supplemented with 10% foetal bovine serum (Gibco), 50 IU/mL penicillin and 50 mg/mL streptomycin (Lonza). For ciPTEC, the medium was additionally supplemented with 5 μg/ml of insulin, 5 μg/ml of transferrin, 5 ng/ml of selenium, 40 pg/ml of tri-iodothyronine, 36 ng/ml of hydrocortisone and 10 ng/ml of EGF (all from Sigma) [26 (link), 27 (link)]. HK-2 and ciPTEC cells were transfected with plasmid vectors using either TransIT-LT1 (Mirus Bio LLC) or jetPRIME (Polyplus Transfection), according to the manufacturer’s instructions, and incubated for 16–20 h before fixation.