Infectious titers of MACS-isolated cells after homogenization by sonication and ribolyzation.

‡Inputs of infectious cell homogenates are expressed as cell number equivalents. Aliquots of 30 µl were inoculated i.c. into groups of six Tga20 mice for mouse bioassay and 300 µl aliquots were layered onto prion-susceptible cells per well for SCEPA, respectively.†Infectious titers were calculated according to the Spearman-Karber method [55] and are expressed as log LD50/g ± SE for bioassay and log TCIU/g ± SE for SCEPA.$Infectious titers were calculated using a GLM with binomial family complementary log-log link and expressed as mean log TCIU/g ± SE of two independent experiments with six technical repeats each.#Controls represent MACS-isolated pDCs and B lymphocytes from spleens 129/Sv×C57BL/6 mice inoculated with 1% (w/v) uninfected CD1 brain homogenates and sacrificed at 30 dpi.*Level of significance for maximum likelihood estimates (GLM) between infectious titers of ribolyzed versus sonicated pDCs as determined by SCEPA.Four 129/Sv×C57BL/6 mice were inoculated i.p. with 100 µl of 1% (w/v) RML and 1% (w/v) uninfected CD1 brain homogenate (control), respectively. At 30 d.p.i spleens were dissected and pDCs and B cells isolated by MACS according to Figure 1. Aliquots of 1×107 cells/ml OFCS, supplemented with protease inhibitors were homogenized by sonication (A) or ribolyzation (B) according to Materials and Methods. To determine infectious titers the cell homogenates were serially diluted 1∶10 and inoculated intracerebrally into Tga20 mice or transferred onto layers of susceptible PK1-2 cells in parallel experiments. Infectious titers were determined by non-parametric statistical analysis for bioassay (Spearman and Karber) and GLM for SCEPA and expressed as log LD50 units/106 cells and log TCIU/106 cells, respectively. A 10−2 dilution of cell homogenates corresponds to 2×105 cell equivalents/ml or 6×103 cell equivalents per 30 µl inoculum for mouse bioassay and 6×104 cell equivalents per 300 µl per well for SCEPA, respectively. Infectious titers represent log mean values ± SE of six independent experiments for SCEPA and log mean values ± SE of a single experiment for mouse bioassay.