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. 2003 Sep 22;100(20):11666–11671. doi: 10.1073/pnas.1834432100

Fig. 3.

Fig. 3.

Dose- and time-dependent response of susceptible and nonsusceptible N2a cells to various dilutions of RML homogenate in the SC assay. (a) Representative wells of an ELISPOT plate showing spots given by N2aPD88 cells exposed to the indicated dilutions of RML strain or to medium (control), as described in experiment 1. (b) Double-logarithmic plot of spot number versus RML strain dilution. Experiment 1 (filled symbols): 20,000 susceptible cells (N2aPD88, circles) and nonsusceptible cells (N2aR33, squares; N2aATCC, triangles) were incubated for 3 days with serial 1:10 dilutions of RML strain (I2424) in OFCS, as indicated. The cells were split three times 1:10, aliquots of 25,000 cells were filtered onto membranes of an ELISPOT plate, and PrPSc-positive cells were determined by the SC assay. Mean values of quadruplicate measurements ± SD are shown. Background values of noninfected cells (19 ± 4 for N2aPD88, 5 ± 1 for N2aR33, and 9 ± 2 for N2aATCC) were subtracted. N2aPD88 cells are ≈3 logs more susceptible than the nonsusceptible cells. Experiment 2 (open symbols): Serial 1:3.3 dilutions of RML strain (I2424, 10–1) into uninfected CD1 brain homogenate (10–1) were diluted 1:1,000 in OFCS, to give a final dilution of total brain homogenate of 10–4 and the dilution of RML strain indicated. Twenty thousand susceptible (N2aPD88, circles) and nonsusceptible cells (N2aR33, squares) were exposed to the samples and processed as in experiment 1. Mean values of six measurements ± SD are shown. Background values of cells incubated with noninfected CD1 homogenate (16 ± 7 for N2aPD88 and 6 ± 6 for N2aR33) were subtracted. In the double-logarithmic plot the relationship between spot number and RML strain dilution is linear from 10–7 to 10–5 (correlation coefficient r = 0.94). The values for the dilutions 10–7 and 3.3 × 10–7 are significantly over background (P = 0.006 and P = 0.0007, respectively; Mann–Whitney U test) but not that for 3.3 × 10–8.(c) Time-dependent increase in the proportion of infected cells. PD88 cells were exposed to a 10–5 (•) or 10–6 (○) dilution of RML strain (I2424) in OFCS for 3 days and split 1:10 every third day. The spot number per 25,000 cells was determined at the times indicated. Mean values of triplicate measurements and SD are shown. Spot numbers were corrected for background values of noninfected cells (16 ± 7). The rate of accrual is initially ≈20–30% per day but levels off as the saturation value of detectable infected cells is approached (≈2,000 per 25,000 cells).