Gm08680

Human skin fibroblast from healthy male donor (Coriell Institute #GM08680 (referred to as control)) and from NPC2 patients (Coriell Institute #GM18455) were purchased from Coriell Institute for Medical Research (NJ, USA). They were cultured in T25 culture flasks, at 37^o C in an atmosphere of 5% CO₂ in complete DMEM culture medium supplemented with 1% glutamax, 1% Penicillin–Streptomycin and 10% or 20% FBS respectively. Primary fibroblasts from NPC1 patients (Coriell Institute #GM03123) were cultured under same conditions, in 15% FBS in EMEM supplemented with 1% Penicillin–Streptomycin. Cells were checked daily and split with trypsin when a confluency of 90% were reached. Prior to fluorescent microscopy, cells were placed on 35 mm microscope dishes with glass bottom (P35G-1.5–50-C, MatTek) (coated with poly-D-lysine for NPC2 diseased cells) and allowed to settle for 48–72 h in their culture medium. All live cell imaging was carried out in M1 buffer containing 150 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 5 mM glucose and 20 mM HEPES (pH 7.4) as described^{59 (link)}. All experiments on cells were carried out in accordance with ethical guidelines and safety regulations defined by the provider Coriell Cell Repositories (www.coriell.org) and the University of Southern Denmark.