Prions are released from scrapie-infected splenic cell cultures <i>ex vivo.</i>

†<p>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.</p><p>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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002538#ppat-1002538-g001" target="_blank">Figure 1</a>. 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.