Fig. 5

Microenvironmental factors influence the fraction of IFNα-producing pDCs. a, b The pDCs were coated with cytokine capture reagents, encapsulated in droplets of varying size (b, scale bar equals 100 µm), and stimulated individually with 50 µg/mL CpG-C for 12 h. The distribution of cells in droplets was measured by manual analysis of microscopic images after production. After staining for viability and surface marker expression, viable cells and TNFα-positive cells were detected via flow cytometry. c Shown is the fraction of cell-containing droplets with exactly one cell; n > = 3. Lines indicate mean, hinges mark interquartile ranges, and whiskers reach to the highest/lowest value that is within 1.5 × interquartile range. d The fraction of IFNα-secreting pDCs was plotted against droplet volume. Dots of the same color indicate cells from the same donor. Gray dots are all originating from different donors. Linear regression was employed to calculate a trend line; n = 24. a, e The pDCs were stimulated in ~92 pL droplets with an increasing fraction of droplets containing more than 1 cell. f Shown is the fraction of IFNα-secreting cells plotted against the fraction of droplets containing more than 1 cell. Two models were generated to explain the observed pattern: (red) two random pDCs co-encapsulated in the same droplet can induce type I IFN production in each other; (blue) rare early type I IFN-producing cells can induce type I IFN production in conventional co-encapsulated cells. The root-square-mean error (RMSE) was calculated for both models to compare the fit to the data; n = 24. g Schematic overview of both models