Isopycnic centrifugation as method for the separation of rat thymocyte subpopulations

Rat thymocytes were separated into five density layers by isopycnic centrifugation on a discontinuous-density Ficoll gradient and each fraction was tested for metabolic functions at different times during a 96-h culture period in various conditions. Immediately after separation the rate of oxygen consumption per unit of cellular volume is virtually the same in all fractions. However, the capacity to synthesize nucleic acids, proteins, purine and pyrimidine nucleotides by the de novo pathway and the activity of the salvage pathway of purine nucleotides is much greater in the low density fractions (fractions 1 and 2). In in vitro cell culture conditions (1) in the absence of mitogens this metabolic activity (which demonstrates a high spontaneous mitotic capacity of cells in the low-density fractions) decays rapidly to a level which is practically negligible after 24 h of culture; (2) in the presence of mitogens alone: PHA, Con A, PWM or PNA, with or without prior neuraminidase treatment, after 24 h an induction of the synthesis of nucleic acids and protein is observed concomitant to the apparition of blast cells. This blastogenesis peaks at 72 h and occurs in fractions 1 and 2 only; (3) in the presence of Con A and 2-mercaptoethanol the blast transformation corresponding to the response of the thymocytes to TCGF is observed in all of the thymocyte fractions and extends beyond 72 h of culture. It remains much more intense in the low-density fractions 1 and 2.