Tracking Flk-1+ cells in vitro and at chronic phase in vivo. A) Tube formation assay in vitro. PKH26 red-labeled Flk-1+ iPS cells were co-cultured with HUVECs for 24 hours on Matrigel. iPS cells (red) were confirmed to be incorporated into network structures. The bar indicates 200 μm. B) Double fluorescence staining of CD31 (green) and PKH26 (red) in ischemic adductor muscles on postoperative day 21. Co-localization is indicated by yellow in the merged images (magnification, ×400; bar indicates 50 μm). Flk-1+ cells (6 × 104 cells) were stained with PKH26 red and then injected into ischemic adductor muscles. Double positive cells (▲) and single positive PKH26 cells(↑) are indicated.