Cultivation of PC12 and hPheo1 Pheochromocytoma Cells

Rat pheochromocytoma cell line, PC12 [13 (link)], and the human progenitor pheochromocytoma cell line, hPheo1 [14 (link)], were generous gifts from Arthur Tischler and Hans K. Ghayee. PC12 cells were cultivated in RPMI-1640 containing 10% horse serum (HS) and 5% fetal calf serum (FCS) on collagen A-coated cell culture dishes. For hPheo1 cells, RPMI-1640, containing 10% FCS and 2 mM Glutamax, was used (detailed experimental procedures and results are summarized in Supplementary Materials). If not stated otherwise, cells were cultivated under normoxic conditions (5% CO₂, 37 °C, and 95% humidity) in a CO₂ incubator (HERAcell, Thermo Scientific). For the simulation of hypoxic conditions, cells were incubated in reduced oxygen partial pressure (≤1% O₂) using an incubator equipped with an oxygen-sensor (Gasboy, Labotect, Göttingen, Germany). All cells were passaged up to 10 times after thawing, and experiments were conducted after passaging the cells at least once. Therefore, cells were trypsinized (trypsin/EDTA; 0.05%/0.02%), diluted with complete medium and counted by using C-CHIPs (Neubauer improved). All cell lines and the subsequently described sub-cell lines were regularly tested to be mycoplasma free (MycoAlert Mycoplasma Detection Kit, Lonza, Basel, Switzerland).