Figure 1.

PBMC derived M1-like macrophages displayed an ischemic phenotype. (a) Flow cytometry analysis and image representation of CD14⁺ monocytes differentiated with GM-CSF alone or in combination with LPS + IFNγ (n = 3). Scale bar 75 μm. (b) Flow cytometry quantification of the M1-like differentiation efficiency upon treatment with GM-CSF, LPS or IFNγ alone, and LPS + IFNγ co-treatment. Data are represented as % of CD64⁺/CD80⁺ differentiated cells for each condition treatment (n = 3). (c) IL-1β and IL-10 protein quantification in the conditioned medium of M0, M1 and M2 macrophages. Protein concentration has been calculated according to the standard curve of each ELISA assay (n = 3). (d) Schematic representation of the IRI protocol applied to M1-like macrophages. (e) Intracellular ATP quantification in M1-like macrophages challenged with cold ischemia at different timepoints. Data are represented as % of ATP in the experimental conditions vs. control (Cn) not induced to ischemia (n = 4). (f) Image representation of M1-like macrophages subjected to full IRI protocol. Scale bar 200 μm. (g) Left panel, quantification of ROS production in M1-like macrophages subjected to full IRI. Data are represented as % of ROS production in experimental samples vs. unperturbed control cells. Statistical significance has been calculated via ordinary one-way ANOVA (Dunnet’s multiple comparisons test, n = 4, * = p < 0.05; ns = not significant). Right panel, gene expression analysis of Romo1 gene on reperfused M1-like macrophages at different timepoints. The comparison has been conducted by using the ∆∆CT method and normalized to GAPDH transcript. Statistical significance has been calculated via ordinary one-way ANOVA (Dunnet’s multiple comparisons test, n = 3, * = p < 0.05; ** = p < 0.005; *** = p < 0.0005).