Fig. 1
Schematic representation of assays work-flow. Optimization of DiR labeling steps. A ECFCs (1·106) were labeled with 6.67 µM DiR, and 2·105 and 1·105 cells (a) were plated after 3 consecutive washes with 1 ml PBS. Also, 3 sets of ECFCs (1·105 each) were labeled with the consecutive supernatant solutions from washing steps (b-d). B DiR- labeled ECFCs (1·106) were washed three times with 10 ml PBS 1X, and 2·105 and 105 cells were plated (a´). Again, sets of 105 ECFCs were labeled with the supernatant solutions and seeded (b´- d´). C Balb-c nude mice were injected with DiR-labeled ECFCs (1·106 in 50 µl), intravenous (IV +) or intramuscularly (IM +), or with 50 µl of the third washing supernatant solution (10 ml PBS each) applied to these cells (IV- and IM-). In vivo scans were performed on day 0 and 1. An ex vivo scan was carried out on day 1. D) CLTI Balb-c nude received 106 DiR-labeled ECFCs (50 µl) intravenously (IV + , n:4) or intramuscularly (IM + , n:4). Also, a DiR residual negative control was included, consisting in CLTI mice receiving via tail vein (IV-, n:1) or intramuscularly (IM-, n:1), 50 µl of 0.2 µM DiR, equivalent to the third supernatant washing solution. An additional mouse was transplanted with 106 unlabeled ECFCs to consider background signal (NC)