The study by Botstein and Sato (1978) investigates the growth of the rat neuroblastoma B104 cell line in a serum-free medium supplemented with insulin, transferrin, progesterone, selenium, and putrescine (N2 medium). The B104 cell line, originating from the central nervous system, was found to proliferate effectively in this serum-free medium, achieving the same saturation density as in medium with 10% fetal calf serum. The addition of each supplement individually had little effect, but in combination, they exhibited a synergistic effect, increasing cell number by 26-fold. The cells in N2 medium showed more extensive neurite extension and retained other differentiated properties, such as action potentials and choline acetyltransferase activity. The study highlights the importance of serum-free media for long-term studies and the potential of using N2 medium to study neuronal cell properties and hormonal responses.The study by Botstein and Sato (1978) investigates the growth of the rat neuroblastoma B104 cell line in a serum-free medium supplemented with insulin, transferrin, progesterone, selenium, and putrescine (N2 medium). The B104 cell line, originating from the central nervous system, was found to proliferate effectively in this serum-free medium, achieving the same saturation density as in medium with 10% fetal calf serum. The addition of each supplement individually had little effect, but in combination, they exhibited a synergistic effect, increasing cell number by 26-fold. The cells in N2 medium showed more extensive neurite extension and retained other differentiated properties, such as action potentials and choline acetyltransferase activity. The study highlights the importance of serum-free media for long-term studies and the potential of using N2 medium to study neuronal cell properties and hormonal responses.