Figure 3. Naïve macrophage response to pro-inflammatory M1 signaling cues is proportional to the stiffness of the culture substrate. (A) Nitrite concentration in cell culture media supernatants from cells cultured on increasingly stiff substrates (n=32). (B) Nitrite concentration in cell culture media supernatants as a function of substrate stiffness and cECM coating age (n=16). (C) Representative immunolabeling of inducible nitric oxide synthase (iNOS) and cell nuclei (DAPI) in Th1 cytokine treated macrophages cultured on 8 kPA gels, 64 kPA gels, or tissue culture plastic each coated with either young or aged cECM. Representative image brightness and contrast adjusted to reduce background and highlight sample differences. TCP= tissue culture plastic. Scale bars = 100um. Data reported represents mean values with boxes representing 25%/75% of mean. Error bars represent standard deviation. ANOVA with Tukey HSD post-hoc analysis. * p<0.05, ** p<0.001.