**4. Results**

Figure 2a shows the gating strategy used for the identification of leukocyte subpopulations, i.e., neutrophils and macrophages. First, we gated on leukocytes based on forward scatter (FSC) and side scatter (SSC) properties and excluded small cell debris. Next, we gated on single cells by plotting the height against the area for forward scatter (FSC-H vs FSC-A), and the width for side scatter against the area for forward scatter (SSC-W vs FSC-A). After doublets exclusion, we distinguished live cells (identified as DAPI negative) CD45.2+ leukocytes. Within live leukocytes we identified CD11b+ and Ly6G+ neutrophils, CD11b+ and F4/80<sup>+</sup> macrophages. The proportion of neutrophils and macrophages were quantified on day 1 and 3 after ligation (Figure 2b). IHC staining demonstrated a localization of neutrophils and macrophages in the perivascular space of growing collateral arteries in the adductor muscle of mice as exemplary shown for day 3 after induction of arteriogenesis by FAL (Figure 2c).

**Figure 2.** Gating strategy of flow cytometry and immunohistological analyses. (**a**) Sequential gating strategy for the identification of neutrophils (CD45.2+CD11b+Ly6G+) and macrophages (CD45.2<sup>+</sup>CD11b<sup>+</sup>F4/80<sup>+</sup>) in the murine adductor muscle containing growing collateral arteries at day 1 after FAL. (**b**) Bar graphs showing the frequencies of neutrophils and macrophages at day 1 and day 3 after FAL. *n* = 3 mice/group, data are represented as mean ± S.E.M., \* *p* < 0.05 from unpaired student's *t*-test. (**c**) Representative immunohistochemical stains demonstrate the presence of neutrophils (Ly6G+) and macrophages (CD68+) (indicated by arrows) in the perivascular space of growing collaterals in the adductor muscle of a mouse 3 days after FAL. Collaterals were stained with an endothelial marker (CD31) and nuclei with DAPI. Scale bar 20 μm.
