Figure 2

Seeding profile of CWD prions from organotypic slice cultures. (A–C) Representative traces from RT-QuIC assay of slices exposed to brain homogenates from normal (NBH) or CWD-infected deer and cultured up to 52 dpi. Each trace comprises 0.5-h time points represented as an average ThT fluorescence from n = 3–4 replicate wells. (A) Seeding activity in CWD-infected slices accelerated rapidly after 21 dpi (n = 3). (B) Lysates prepared from 52-dpi slices were serially diluted 10-fold, ranging from 50 fg to 50 ng. The reactions (n = 4), consisting of 5 µL of these dilutions as seed, revealed optimal seeding doses ranging between 50 pg to 50 ng. (C) Endpoint 10-fold serial dilution analysis (n = 4) of the CWD deer BH used as inoculum. (D) Quantification of seeding activity from different time points in the CWD slice culture model. The median seeding dose (SD₅₀) is defined as the amount giving sufficiently enhanced ThT fluorescence in half of the replicate wells. The Spearman-Kärber estimate of the SD₅₀, plotted against days post-inoculation of the slice culture, shows prion titers progressively increase to a maximum at 52 dpi.