Figure 2. The immune response induced by FBS-containing hydrogels is characterized by increased local concentrations of cytokines associated with type 2 immunity.

Day 7 hydrogel explants were digested and a Luminex bead-based multiplex ELISA was run to analyze the local cytokine milieu. (a) Heatmap with two-way hierarchical clustering using Ward’s method with Euclidian distances shows distinct clustering of FBS-containing dorsal hydrogels based on local cytokine secretion. Scale bar represents data that has been log transformed, unit variance scaled, and mean centered. (b) Principal component analysis of cytokines within dorsal hydrogels. Principal component 1 and principal component 2 are displayed on the X- and Y-axes and explain 36.1% and 20.8% of the variance, respectively. 95% prediction ellipses are also plotted. (c) Cytokines with significantly different local concentrations, as well as key type 1 and type 2 immune response cytokines with no significant differences in concentrations (n=6 samples per group). Red, dashed line indicates the limit of detection of the Luminex MAGPIX System. A one-way ANOVA was used to detect statistical differences followed by Tukey’s multiple comparisons test for IL-4, Eotaxin, IL-9, IL-25, and IL-33. For all other cytokines, a Kruskal-Wallis test was used followed by Dunn’s multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.