Ivis spectrum small animal in vivo imaging system

Tetrakis (4-carboxyphenyl) porphine was encapsulated into the composite nanosystems as a fluorescent probe. A mouse wound infection model was established to test the targeting delivery performance of the composite nanosystems. Briefly, the nude mice were anesthetized with ether cotton balls and the epidermis was cut on the right hindlimb by surgical scissors to form a 5 mm diameter wound. Subsequently, S. aureus was injected into the wound (10⁷ CFU/mL, 100 μL). After infection for 24 h, the same amount of free fluorescent probe and composite nanosystems loaded fluorescent probe were injected intravenously (n = 3). After medication, the fluorescence intensity at the infection site was observed at a given time (6, 12, 24, and 48 h) by the IVIS Spectrum small animal in vivo imaging system (PerkinElmer, USA). At the end of the experiment, the fluorescence of the viscera was measured after the mice was slaughtered.

Ad-SVF and ad-SVF spheroids were labelled with both the CellTracker™ CM-DiI and DiI-C₁₈(5)-DS (Molecular Probes) before transplantation according to the manufacturer’s instructions. Briefly, CM-DiI (2 μg/mL) and DiI-C₁₈(5)-DS (2 μg/mL) were performed to incubate cells for 5 min at 37 °C and following 15 min at 4 °C. Labelled ad-SVF or ad-SVF spheroids were injected to bladder and detected by IVIS Spectrum small-animal in vivo imaging system (PerkinElmer) and fluorescence microscope after 4 weeks.

EPCs were labelled with both the CellTracker™ CM‐Dil and Dil‐C₁₈(5)‐DS (Molecular Probes) before tail vein injection according to the manufacturer's instruction. Briefly, EPCs were incubated with CM‐Dil (2 μg/mL) and Dil‐C₁₈(5)‐DS (2 μg/mL) for 5 minutes at 37°C and following 15 minutes at 4°C, and washed twice before tail vein injection. The labelling efficiency of CM‐Dil and Dil‐C₁₈(5)‐DS was verified using fluorescence microscope and FACSCalibur, respectively. Labelled EPCs (2 × 10⁶) were injected by using a previously described protocol with minor modifications,²⁵ and their fate and distribution in kidneys were investigated by IVIS Spectrum small‐animal in vivo imaging system (PerkinElmer) and fluorescence microscope. Briefly, the kidneys of rats injected with EPCs were collected 24 hours after reperfusion. Following a detection by the in vivo imaging system, the kidneys were cryopreserved in OCT compound (Tissue‐Tek), followed by preparation of 5‐μm cryosections, fixation in acetone for 2 minutes and blocking for 30 minutes in PBS + 3% bovine serum albumin (BSA, Biofroxx). Then, sections were incubated with anti‐CD133 (Abcam) and DAPI. Subsequently, fluorescence microscope (Olympus) was adopted to monitor CM‐Dil (red) and CD133 (green) double‐positive cells.