Fig. 3

hADSCs injection in the ischemic muscles increased the infiltration of the proangiogenic M2 macrophages. Immunofluorescence analyzes in collected muscles 3 (a, b, c, d) and 7 (e, f, g, h) days after the ligation of the artery and the administration of hADSC (d, h), mADSC (c, g), mBM-MSC (b, f), and control (PBS⁻) (a, e). The representative images of M2 macrophages (F4/80⁺ (red) / CD206⁺ (green)). The images show that after the hADSC injection into ischemic muscle the infiltration of the proangiogenic macrophages M2 was observed on the 3rd day after the surgery and it increased at day 7. After the administration of mBM-MSC and PBS⁻, the significant M2 macrophage infiltration was not observed. However, after the administration of mADSC an increase in macrophage infiltration was observed on day 7. The graph of the total area of F4/80 macrophages in collected muscles on the 3rd and 7th day after the surgery (I). The total area occupied by F4/80 macrophages was calculated using ImageJ software in 10 fields of view at a magnification of 40 × (n = 5; 5 muscles per group were analyzed; 10 pictures of each muscle were taken). Using the flow cytometry 7 days after the artery ligation and administration of the hADSCs, mADSCs, and PBS⁻ in collected muscles the number of M1 (defined as 7-AAD⁻/CD45⁺/F4/80⁺/ CD86⁺) and M2 (defined as 7-AAD⁻/CD45⁺/F4/80⁺/CD206⁺) macrophages was evaluated (j). In all researched groups we observed a greater number of M2 macrophages in comparison to M1 macrophages. The largest number of M2 macrophages was found in muscles after the administration of hADSCs: six times more than mADSCs group and sixty times more than PBS⁻ group. The ratio of M2 to M1 macrophages was 686 in hADSCs group, 824 in mADSCs group and 364 in PBS⁻ group (n = 5). **p < 0.001 evaluated with Kruskal-Wallis one-way analysis of variance and multiple comparison of mean ranks for all groups. ***p < 0.0005 by the ANOVA followed by the Tukey’s post hoc test. Nuclei stained with DAPI (blue), scale bars = 50 μm