Age-related factors in cyclosporine-induced syngeneic graft-versus-host disease: regulatory role of marrow-derived T lymphocytes

Briefly, 0.4-1 x 10' bone marrow cells suspended in 5-10 ml of elutriation medium (0 .9% saline, 0.5% BSA, 0.3 mM EDTA, pH 7.20) were injected into the inlet stream leading into theJ6M centrifuge equipped with a JE-6B elutriator rotor and standard chamber (Beckman Instruments, Palo Alto, CA). The cells were loaded into the chamber at a flow rate of 15 ml/min, a rotor speed of 3,000 RPM (900 g), and a temperature of 18°C. Rotor speed was held constant and the cells were eluted by changing the flow rate. In the case of rat bone marrow (17), lymphocytes were purged at a flow rate of 25 ml/min (400-ml collection), and intermediate sized cells at 29 ml/min (200 ml). Cells still remaining in the elutriator chamber were collected by continuing medium flow after stopping the rotor, and were designated the rotor-off (R/0) fraction . The cells were then washed and resuspended in RPMI 1640 .