Mammalian cell culture

Primary human skin fibroblasts (line C688) were obtained from the Department of Metabolic Diseases, The Children’s Memorial Health Institute in Warsaw, Poland [30]. Ganglioside GM₁ molecules, receptors for CTX, are one component of fibroblast membranes. C688 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma, USA) with 10% fetal bovine serum (FBS, Gibco, South America), 100 units/mL penicillin and 100 μg/mL streptomycin (Sigma, USA) at 37 °C in a humidified atmosphere containing 5% CO₂, on 100 mm tissue-culture treated dishes (Corning BD, USA) until 80–90% confluence. To dissociate the adherent cells, dishes were rinsed with phosphate buffered saline solution pH 7.5 (PBS, 10 mM Na₂HPO₄, 1.76 mM KH₂PO₄, 2.7 mM KCl, 136 mM NaCl) and incubated with 2 mL (0.5 mg/mL) of porcine trypsin (Sigma, USA) for 7 min at 37 °C. Cells were collected by centrifugation (500 g for 3 min), resuspended in fresh medium and counted under a light microscope (Zeiss Observer Z1, Germany) using a Bürker chamber.