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Fig. 2 | Stem Cell Research & Therapy

Fig. 2

From: Assessment of endothelial colony forming cells delivery routes in a murine model of critical limb threatening ischemia using an optimized cell tracking approach

Fig. 2

Determination of DiR residual staining. A Representative scans from calibration curve on days 1 and 3. ECFCs were incubated with 6.67 µM DiR, plated in ½ dilutions (from 200.000 cells to 781) and scanned on days 1, 2 and 3. B Standard curves (DiR intensity vs cell number) and linear equations obtained for days 1 and 3, corresponding to the averaged values of three independent standard curves performed. C Representative images obtained for DiR pre-labeled ECFCs (2·105 and 1·105) (top wells) and ECFCs (1·105) incubated with the 3 consecutive washing solutions (1 ml PBS each) derived from the cells in the upper wells (bottom wells). D Scans from Labeled-ECFCs (2·105 and 1·105) (top) and ECFCs (1·105) incubated with the remaining washing solutions (10 ml PBX 1X) from the, (bottom). E ECFCs (1·105) were incubated with decreasing amounts of DiR (from 3,34 to 1.6·10–3 µM DiR, serial dilutions 1/2) F Standard curve and linear equation obtained after the incubation of ECFCs with DiR serial dilutions, corresponding to the averaged values of three independent standard curves performed. G Scanned plate with ECFCs (1·105), unlabeled (DiR negative control), incubated with 6,67 µM DiR without washing or pre-incubated with 6.67 µM DiR after 3 washes (top row). ECFCs incubated with the supernatant from 3 consecutive washing solutions (lower row). H In vivo scans on day 0 (left) and day 1 (right) after administration of 0.2 µM DiR either IV (IV-) or IM (IM-), or administration of 106 labeled ECFCs (6,67 µM DiR, IV + and IM +). I Ex vivo scans of spleen, kidneys, lungs, liver) and hind limbs, one day after injecting either DiR-labeled ECFCs or 0.2 µM DiR. A Negative control (with 106 unlabeled ECFCs), was also included to discard background signals

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