Advancebio sec 5 column

EV samples were prepared using ultracentrifugation as described above. All experiments were performed using high-performance liquid chromatography (HPLC) instrument Agilent 1100 with an AdvanceBio SEC-5 column (300 Å 2.7 μm, 7.8 × 300 mm, Agilent) with mobile phase PBS (pH 7.4, 0.5 mL/minute), with UV absorbance (DAD) at 220 nm and 280 nm and fluorescence detection (FLD) at 655 nm, and acquired using Agilent software (ChemStation Rev. A.10.02). Representative aliquots containing 1 × 10⁹–1 × 10¹⁰ EV and/or 0.5-2.0 μL Qd655-SAV were used to measure the retention time of the unlabeled EV population, unconjugated Qd655-SAV, and CD9biotin-Qd655-SAV labeled EV. Based on these retention times, EV fraction collection was scheduled to maximize capture of labeled EV with minimal contamination of free Qd. A second fraction was timed to capture the majority of free Qd. Primary labeling of EV with CD9-biotin was performed as described above. Samples were prepared for HPLC separation using 5 × 10⁹ primary labeled EV (counted on NTA after the primary-Ab wash) and 2 μL Qd655-SAV. Here, excess secondary Ab (compared to standard labeling protocol) was used to allow for greater visualization and recovery of Qd in the HPLC chromatogram and fraction analysis. NTA was performed immediately on the collected fractions.