Substrate elasticity significantly affects association of Tf with REF52 cells after a 10-minute but not 1-hour incubation but does not have a significant effect on CTb association.

<p>(A) Schematic representation of experimental setup used to quantify cell association of fluorescent markers. Cells on PEG hydrogels of variable elasticity were incubated for a specified duration with fluorescent markers, washed to remove non-associated markers, detached using trypsin and analyzed using flow cytometry. Histograms of cell-associated fluorescence were acquired after gating live cells. (B) Histograms of a typical experiment showing efficient internalization of Tf by cells on hydrogels compared to negative controls and exhibiting a narrow Gaussian distribution. Tf association with REF52 cells was similar on soft, intermediate and stiff substrates after 1 hour incubation (C) but showed a significant lower normalized MFI on softer gels at the shorter time point of 10 minutes (D). Dot plots and mean values are presented, with each dot representing an independent experiment. (E) Epifluorescence microscopy images of REF52 cells on substrates with intermediate stiffness incubated 1 hour with Alexa Fluor 568-labeled Tf and stained for plasma membrane (WGA) confirmed that uptake of Tf by REF52 cells was uniform among the cell population and further showed that Tf localized in a perinuclear region of the cells. Scale bar: 100 µm (F) Histograms of a typical experiment of CTb association with REF52 cells showing a broad distribution of intensity value/cell. Normalized MFI values of CTb association were similar on soft, intermediate and stiff substrates after both 1 hour (G) or 10 minute (H) incubation with CTb. Dot plots and mean values are presented with each dot representing an independent experiment. (I) Epifluorescence microscopy images of REF52 cells on substrates with intermediate stiffness incubated 1 hour with Alexa Fluor 568-labeled CTb and stained for plasma membrane (WGA) showed a heterogeneous population of cells in respect to CTb internalization. Scale bar: 100 µm.