Xenogen ivis spectrum noninvasive quantitative molecular imaging system

To further track the bioactivity of the implanted CSPCs in vivo, CM-DiI (Molecular Probes, Eugene, OR, USA) was used as a non-targeted probe for CSPCs before transplantation. All procedures were performed as previously described [34 (link)]. The applied label, CM-Dil (excitation: 553 nm; emission: 570 nm), for CSPCs was monitored by red fluorescence in the defect zones at 4 and 12 weeks after implantation. The Xenogen IVIS^® Spectrum Noninvasive Quantitative Molecular Imaging System (PerkinElmer, Waltham, MA, USA) was used for optical imaging for cell tracking.

To track the implanted EPCs in vivo, they were stained with chloromethyl-benzamidodialkylcarbocyanine (CM-DiI; Molecular Probes, Carlsbad, CA, USA) [53 (link)] before transplantation. The red fluorescence from CM-Dil was visible in the defect zones, and the eluent was monitored by fluorescence at excitation at 553 nm/emission 570 nm using Xenogen IVIS^® Spectrum Noninvasive Quantitative Molecular Imaging System (PerkinElmer Inc., Waltham, MA) 4 and 12 weeks after implantation.

The indocyanine green (ICG) solution used in this study was prepared by dissolving ICG powder (Carbosynth Limited, UK) in alpha minimum essential medium (αMEM, Gibco) to give a stock solution of 2.5 mg/mL. For ICG labeling, 1 × 10⁶ ASCs were incubated at 0.5 mg/ml of ICG solution for 30 min at 37°C. After labeling, the cells were washed three times with DPBS and further cultured in complete αMEM. ASCs were injected directly into the compressed sciatic nerve, and each rat was imaged every 2 days for 9 days until the fluorescence signal matched that of the PBS control. In vivo fluorescence imaging was performed with a Xenogen IVIS^® Spectrum Noninvasive Quantitative Molecular Imaging System (PerkinElmer Inc. Waltham, MA). All images were acquired using an excitation wavelength of 745 nm and an emission wavelength of 820 nm. Following the acquisition, all images were normalized to the units of the average efficiency and displayed on the same scale of fluorescence intensity. Bioluminescence from the region of interest was manually delineated, and the data were expressed as photon fluxes (photons/s/cm²/steradian). The bioluminescence data were collected and analyzed using the IVIS^® Living Image System.