Cardiogreen (Foxgreen)

Protocol for Cardiogreen (Foxgreen) uptake assay in cell:

Cardiogreen (Foxgreen, indocyanine green) is a non-toxic biological dye, it only can be taken up by hepatocytes after intravenous injection, after which it is secreted by hepatocytes. When cultured with serum-free medium, hepatocytes take up Cardiogreen, and the cytoplasm appears green. After restoration of serum, Cardiogreen is secreted out of the cytoplasm.

1. Add Cardiogreen to serum-free hepatocyte medium with a final concentration of 1 mg/mL and cells are cultured with the medium for 1 hour at 37℃.

2. Remove serum-free medium, and wash cells with PBS for three times.

3. Cells are examined and imaged with a microscope.

Protocol for Imaging the urinary pathways in mice by liposomal Cardiogreen ^[1]:

0-0.8 μM Cardiogreen was incubated with 1% liposomes and 20 μM or 200 μM pyrene for evaluation of polarity and fluidity, respectively. Fluorescence spectra were measured using Fluorescence Spectrophotometer at excitation wavelength 335 nm.

1. Mice were shaved under isoflurane (1%-2%, v/v) anesthesia and underwent baseline scans using an in vivo imaging system.
2. Two hundred microliters of 8 mg/kg Phospholipon 50 Cardiogreen liposomes or the free compound in 2 mM sucrose buffer (phosphate buffer pH 7, with 9.3% sucrose) was injected into the tail vein and the mice were repetitively scanned over a time period of up to 120 min.
3. For ex vivo studies, animals were treated as described above and sacrificed under anesthesia by cervical dislocation, 10 min post injection.
4. Cardiac blood was collected in heparinized 96-well plates and tissues were harvested. Along the collection procedure, tissue and blood samples were protected from light and kept on ice.
5. Immediately thereafter, blood and tissues were scanned by a biomolecular imager.

Protocol for studies of the accumulation of liposomal liposomal Cardiogreen in the urinary system:

1. Liposomal or free Cardiogreen (8 mg/kg). was injected into the tail vein, and 3 min later, mice were treated with high dose pentobarbital administered intraperitoneally.
2. The overall time of active circulation after the Cardiogreen injection was about 10 min.
3. Thereafter, mice were subjected to midline laparotomy with full exposure of the retroperitoneum.
4. The ureters were identified in their origin from the kidneys, and distally clipped or loosely tied for identification.
5. The mice were also hysterectomyzed to ease the urinary bladder identification.
6. Subsequently, the animals were scanned by biomolecular imager.

This protocol only provides a guideline, and should be modified according to your specific needs.

References:

[1].Portnoy E, Nizri E, et,al. Imaging the urinary pathways in mice by liposomal indocyanine green. Nanomedicine. 2015 Jul;11(5):1057-64. doi: 10.1016/j.nano.2015.02.019. Epub 2015 Mar 16. PMID: 25791809.