Iodogen

The CD206-targeting NIRF probe was generated using a previously described method 16 (link). Briefly, anti-CD206 antibody (αCD206, clone C068C2, IgG2a; Biolegend, San Diego, CA) was mixed with Dylight755-N-hydroxysuccinimide (NHS) ester (Pierce, Rockford, IL) in sodium bicarbonate buffer (pH 8.4) at a 1:10 molar ratio. After incubation for 12 h at 4°C, the mixture was purified using a PD-10 desalting column (GE Healthcare, Piscataway, NJ). The degree of labeling (dye/protein molar ratio) of Dylight755-αCD206 (Dye-αCD206) was 6:1, as detected using an ultraviolet spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Dylight755-labeled isotype-matched control rat IgG (Dye-IgG) was synthesized as a control using the same method.
The CD206-targeting radiotracer was generated by radiolabeling anti-CD206 antibody (100 µg) with 185 MBq Na¹²⁵I using a previously described method 24 (link). Briefly, 100 µg of antibody dissolved in 0.2 M PBS (pH 7.4) was mixed with 185 MBq of Na¹²⁵I in a vial pre-coated with Iodogen (Sigma, St. Louis, MO). After incubating at room temperature for 10 min, the product, ¹²⁵I-αCD206, was purified using a PD-10 desalting column. The radiochemical purity of ¹²⁵I-αCD206 was >98%. ¹²⁵I-labeled isotype-matched IgG (¹²⁵I-IgG) was also prepared using the same method.

For expression studies, apoA5 was labeled with ¹²⁵I using Iodogen (Sigma-Aldrich; Merck KGaA) according to the manufacturer's instructions. Differentiated 3T3-L1 adipocytes were pulsed with ¹²⁵I-labeled apoA5 (~2×10⁶ cpm/well) in serum-free medium for 2 h at 37°C. At the end of the pulse, the medium was aspirated and cells were washed three times with ice-cold PBS, and incubated with heparin (10 mg/ml in PBS; Sigma-Aldrich; Merck KGaA) for 3 min at room temperature, and then washed three times with PBS. Fresh medium was added, and cells were chased for various time periods (0, 1, 2, 4, 6 and 24 h) at 37°C. At the end of the chase period, the medium was collected and cells were treated with heparin as described above. This heparin wash was added to the collected medium which was then precipitated with 13% trichloroacetic acid to estimate degradation of apoA5. Following heparin treatment, cells were collected in 0.1 M NaOH. Total cell protein was determined using the bicinchoninic acid assay (Novagen; Merck KGaA). Radioactivity (cpm) measured by gamma-ray counter was normalized to cell protein, and data are expressed as total cpm/mg protein at each time-point.