Persistence of Labeled Bone Marrow-Derived MSCs in Ischemic Limbs

To examine the persistence of bone marrow-derived MSC in the hindlimb ischemia model in BALB/c nude mice, the cells were stained with Vybrant™ CM-DiI Cell-Labeling Solution (Thermo Fisher Scientific, Waltham, USA) as described before [22 (link)], prior to injecting the cells into the animals. Five million viable cells were resuspended in 50 μl PlasmaLyte A and injected i.m in both ischemic and normal limbs. Evaluation of signal intensity from CM-DiI-labeled cells was performed after cell injection on days 1, 3, 6, 11, 14, 21, and 28 using the In Vivo Imaging System (IVIS) Xtreme Imaging System (Bruker, Massachusetts, USA). The images were quantified using Carestream molecular imaging software. As per the IVIS Xtreme Imaging System guidelines, all the animals were imaged before the cell injection to normalize autofluorescence. Average pixel intensity from each group was calculated from the individual animal’s pixel area intensity.