Constitutive and basal secretion from the endocrine cell line, AtT-20

Variants of ART-20 cells, HYA.15.6 and HYA.15.10, were isolated as slow growing clones that survived several rounds of drug selection during a somatic cell hybridization procedure (Kohler and Milstein, 1976; Margulies et al., 1976) between ART-20 F2 ceils (HAT resistant, ouabain sensitive) and the mouse myeloma, 4TOO1 (HAT sensitive, ouabain resistant). Equal numbers of both cell types were mixed in suspension centrifuged at low speed (2,000 g) to form a pellet. The cells were resuspended in serum-free DME containing 35% polyethylene glycol 4,000 (PEG), pH 8.2, incubated for 90 s and then immediately diluted with 40 mi of fresh medium. Cells were removed from the PEG by low-speed centrifugation and then resuspended in DME containing 10% FCS, HAT, and 2.5 mM oubain. The selection medium was changed every 48 h until >99 % of the cells were killed. Slower growing clones exhibiting the qualities described in the results were recovered and subcloned additional times to ensure their homogeneity.