**3. Methods**

### *3.1. Reagent Preparation*

For flow cytometric cell analysis, the following reagents were prepared: FACS buffer, enzyme solution, and three gradient Percoll solutions.

The FACS buffer was prepared as a solution of PBS, 1% fetal bovine serum, 2.5 mM EDTA and 0.02% sodium azide.

Two-fold concentrated enzyme solution was prepared using a solution of collagenase IV and DNase I at a concentration of 400 U/ml (collagenase IV) and 0.4 mg/mL (DNase I) in RPMI-1640, respectively.

For isotonic Percoll stock solution, 45 mL Percoll and 5 mL 10× PBS were mixed. The solution was then prepared in three concentrations (70%, 37% and 30%). For 70% and 30% Percoll, the prepared Percoll stock solution was diluted with Hank's Balanced Salt solution with phenol red. For 37% Percoll, the prepared Percoll stock solution was diluted with PBS. Different diluents were used for easy recognition of phases.

### *3.2. Surgical Procedure for Artery Ligation and Tissue Collection*

The murine hindlimb ischemia model is a widely used mouse model to study arteriogenesis in detail [7]. The femoral artery of the right leg is ligated, while the left leg is sham operated and serves as an internal control [8]. After femoral artery ligation (FAL), the blood flow is redirected into preexisting arteriolar connections, which thereupon experience increased shear stress [9,10], and results in perivascular recruitment of leukocytes supplying growth factors and cytokines to the growing vessels [5,6].

Depending on the experimental setup, the muscle tissue, in which collaterals are located, can be removed at various time points after arterial occlusion. To analyze the numbers and subpopulations of recruited leukocytes, we have chosen two time points: day 1 and day 3 after femoral artery ligation. For this purpose, the hindlimbs of the mice were perfused with FACS buffer via an aortic catheter (inserted distal to the outlet of the renal arteries). Thereafter, the adductor muscle of the mouse was removed along the dashed line as shown in Figure 1, whereby the femoral artery (FA) and profunda femoris artery (PA) were omitted from isolation. The muscle section was immediately rinsed with FACS buffer and further processed on ice.

**Figure 1.** Tissue sampling on the right hindlimb of a C57BL/6J mouse. The part of the M. adductor containing the collaterals (arrows) was extracted along the dashed line. The collaterals connect the profunda femoris artery (PA) to the femoral artery (FA). The epigastric artery (EA) serves as orientation for the ligation. Scale bar 1 mm.

#### *3.3. Stepwise Preparation of a Single Cell Suspension from Collected Tissues*

To prepare a single cell suspension for flow cytometric analysis, collected tissue samples were processed using the following protocol:


### *3.4. Cell Staining for Flow Cytometry*

For cell staining, the single cell suspension that was prepared from collected tissues was processed using the following protocol:

