Primary human adrenal culture cells were isolated as described in Materials and Methods, and plated at a density of 300,000 cells/well in 24-well dishes. Cells were treated with/without ACTH (10 nM) for 48 h before harvest. The concentration of steroids in the medium was measured by LC-MS/MS and normalized to the amount of protein. The amount of four major steroids produced by AA cells, cortisol, corticosterone, 11-deoxycortisol and 11OHA are shown in the graphs with each line representing an individual, independent experiment.