Uflc hplc system

Commercially available compounds were used without further purification unless otherwise stated. BODIPY-FL-C₁₆ was purchased from Thermo Fischer Scientific (99%) (Netherlands). 1,3-diolein was purchased from Sigma Aldrich (≥ 99%). DMEM/F-12 was purchased from ThermoFischer (Waltham, MA).
All HPLC purifications (1.0 mL/min, solvent A; 0.1% TFA in water, solvent B; CH₃CN, 50°C) were performed on a Shimadzu UFLC HPLC system equipped with a DGU-20A₅ degasser, a SPD-M20A UV detector, a LC-20AT pump system, a CBM-20A communication BUS module, a CTO-20AC column oven, and a Scan-RAM radio-TLC/HPLC-detector from LabLogic using an Aeris™Widepore column (XB-C18, 3.6 μm, 4.6 mm × 250 mm) for the BDP-FA or an Aeris™Widepore column (C4, 3.6 μm, 4.6 mm × 250 mm) for the Bodipy-triglyceride (BDP-TG). ESI-MS was performed on a Applied Biosystems SCIEX API 150 EX electrospray ionization quadrupole (ESI-Q) mass spectrometer with the method of McAnoy et al. [34 (link)]. Briefly, 0.1M aqueous ammonium acetate solution was added to the probe to observe the ammonium salt in the MS.
¹H-NMR spectra were carried out on a Bruker UltrashieldTH 400 plus at 400 MHz. Tol-d₈ was used as solvent with TMS as internal standard. Chemical shifts are reported in parts per million (ppm) relative to the internal standard.

An amount of 50 µg of enzymatically depolymerized HS sample was separated by strong anion exchange chromatography on a Shimadzu UFLC- HPLC system connected to a UV-VIS detector (20AD, Kyoto, Japan), as described earlier [49 (link)]. All preparations except for the tumor lobe were prepared thrice, and SAX was measured once for each preparation using a ProPac PA1 analytical LC column (Thermo Fisher, Waltham, MA, USA). Because the tumor lobe material was limited, only a single preparation could be used for disaccharide composition analysis. cHS 1 and 2 from Iduron (Cheshire, UK) and Celsus (Cincinnati, OH, USA) was depolymerized once, and disaccharide composition was in the usual obtained range (data not shown). For relative quantification of HS disaccharide composition, the obtained AUCs were compared with AUCs of commercially available HS disaccharide standards (Iduron, Cheshire, UK). Therefore, AUC from the standards were divided by the applied mol and using these the AUC from the samples were back-calculated for their mol, and further relative abundancy was calculated by dividing through the mol sum of the different obtained disaccharide specimens. The mean of three different preparations was calculated by adding the scores together and then dividing by the number of scores.

Unless otherwise stated, all chemicals and solvents were used without further purification. Water used for this study was ultrapure (> 18.2 MΩcm⁻¹ at 25 °C). Phosphate-buffered saline (PBS) as well as cell growth medium was purchased from the Media Preparation Facility at Memorial Sloan Kettering Cancer Center (MSK) (New York, NY). Humanized monoclonal antibody recognizing FRα and M9346A, as well as antibody-drug-conjugate IMGN853 were provided by ImmunoGen, Inc. (Waltham, MA) and further purified via a PD10 desalting column (GE Healthcare). Concentrations of solutions containing antibody were determined by using a NanoDrop™ 2000 spectrophotometer from Thermo Fisher Scientific (Waltham, MA). The bifunctional chelator p-isothiocyanatobenzyl-desferrioxamine (DFO-Bz-NCS) was purchased from Macrocycles (Plano, TX). ⁸⁹Zr-oxalate was acquired from 3D Imaging, LLC (Maumelle, AR). HPLC reactions (1.0 mL/min, phosphate-buffered saline) were performed on a Shimadzu UFLC HPLC system equipped with a DGU-20A degasser, a SPD-M20A UV detector, a LC-20AB pump system, and a CBM-20A communication BUS module using a size exclusion column (GE Superdex™ 200, 10/300 GL).