Prions are released from scrapie-infected splenic cell cultures ex vivo.

†Control incubations were performed in basal medium at atmospheric (atm.) CO2 and 37°C. For dendritic cell cultures Triton X-100 was added to a final concentration of 0.01% in basal medium.Fifteen 129Sv×C57BL/6 were inoculated i.p. with 100 µl RML I6200 and culled at 60 d.p.i. Splenic cell types were isolated by Collagenase perfusion according to the experimental procedure depicted in Figure 1. The levels of cellular infectivity were determined after MACS isolation. MACS-isolated B and T lymphocytes were then cultured at a concentration of 1×106/ml in basal medium (IMDM medium, 10% FBS) in absence or presence of LPS (50 µg/ml) and IL-4 (10 ng/ml). DCs were cultured in basal medium, supplemented with 200 ng GM-CSF. To remove cells and debris the conditioned medium was collected after 36 h of culture, centrifuged at 300× g for 10 min, 5,000× g for 15 min and 10,000× g for 30 min and the supernatant was collected at each of the sequential centrifugations. The supernatant was then centrifuged for 2 h at 100,000× g and resuspended in PBS, serially diluted and infectious titers were determined using SCEPA. The detection limit for SCEPA was 0.1 TCIU/Mio cells. Mean values ± SE of three independent experiments are shown. Data from two independent experiments are shown for the release of infectivity from DCs.