Figure 5.

Substrate stiffness affects mDC adhesive behaviour and viability, but not motility. (a) Representative brightfield images taken with a 40x objective of mDCs in culture on PAA substrates with stiffness of 2, 12 or 50 kPa after 1 day of maturation by addition of IL-4, GM-CSF, IL-1β, TNF-α, IL-6 and PGE₂ (total 5 days of culture). (b) Cell clump size (in µm²) in mDC cell culture on PAA substrates with stiffness of 2, 12 or 50 kPa after 1 day of maturation (total 5 days of culture). More than 40 clumps in more than 8 fields of view per condition, were measured in brightfield images of 2 donors using in FIJI ImageJ. Bars represent mean with SEM. Statistical significance was tested with one-way ANOVA. (c) Cell velocity (in µm/min) of mDCs on PAA substrates with stiffness of 2, 12 or 50 kPa after 1 day of maturation (total 5 days of culture). Cell velocity was determined by manually tracking >30 single cells per condition over 1 hour time period in brightfield images of 2 donors, using the Manual Tracking plugin in FIJI ImageJ. Bars represent mean with SEM. (d) Viability of day 2 mDCs (n = 8 donors) conditioned on PAA substrates of 2, 12, or 50 kPa, represented as the eFluor780-negative fraction of cell population measured with flow cytometry. Bars represent mean with SEM. Statistical significance was tested with repeated measures ANOVA. **p < 0.01; ***p < 0.001.