Cell immunostaining and flow cytometry

Immunostaining followed by flow cytometry was performed to determine Pgp specificity of DOTA-Pab-IR800. Briefly, 3T3-MDR1 and 3T3 cells were cultured overnight. After washed with PBS, the cells were detached using 0.25% Trypsin, 0.1% EDTA (Corning Inc.) and suspended in PBS buffer. Then, 1 × 10⁶ of live cells were first blocked using 10% goat serum at room temperature for 10 min, and secondarily stained with 10 μg/ml of DOTA-Pab-IR800 or PBS at 4 °C for 30 min. Cell-associated fluorescence was detected on an LSRFortessa flow cytometer (BD Bioscience, CA, USA) and ten thousand cell events were analyzed with FlowJo software (FLOWJO LLC, OR, USA). Measurement of surface Pgp expression in NCI/ADR-RES and OVCAR8 cells was carried out by immunostaining with a PE-labeled anti-Pgp antibody (BD Biosciences) followed by flow cytometry.