Figure 4.

Detection limit and quantitation limit. (A) Dose-dependent inhibition of T cell proliferation by MSCs. PBMCs and MSCs were co-cultured in different ratios to define the lowest MSC concentration for which the inhibition of proliferation can still be detected. Based on the 1:0 ratio, a LOB of 6.8% and a LOD of 13.6% were determined and are highlighted by medium and light grey areas. Mean values for n = 30 (1:1, 1:0.2, 1:0.1, 1:0) or n = 7 (1:0.05) are indicated above the plot. (B) Number of detectable T cell generations with respect to MSC co-incubation at different ratios. The LOB at 5.4 generations and the LOD at 4.3 generations were calculated based on T cell generations in the absence of MSCs and are highlighted within the plot in light and medium grey (n = 30 and n = 7 for 1:0.05). (C) Determination of the LLOQ for one daughter generation. Anti-CD3 and anti-CD28 co-stimulated cells were spiked into unstimulated cells as a negative control for an undivided generation. A dilution series was performed, and the calculated value of divided PBMCs was plotted against the number of detected events (measured value). Data were obtained in three independent experiments. Circles, squares and triangles represent data obtained within one titration experiment, each. Values below the LOD of 97 events and the LOB of 62 events are highlighted in light and medium grey, respectively, and the LLOQ of 100 events is indicated by the dotted lines. The dataset was analysed by computing the Spearman correlation coefficient (r = 0.9392, p < 0.0001).