Clone poly4053

To analyze the expression levels of ABCC3, 4 × 10⁵ cells were counted using a Neubauer chamber using Trypan Blue Solution 0.4% (Fisher Scientific). After incubation in blocking and permeabilizing buffer (PBS/saponin 0.1%/FBS 5%) for 15 min at 4 °C, cells were stained with anti-ABCC3 monoclonal antibody (mAb, 1:150; clone M3II-9; Fisher Scientific) and APC goat anti-mouse IgG antibody (1:1500; clone Poly4053; Biolegend) for 20 min at 4 °C. To stain with nanobodies targeting ABCC3, cells were detached with 10 mM EDTA (Fisher Scientific). Upon incubation in blocking buffer (PBS/1% BSA) for 15 min at 4 °C, cells were stained with 5 µg of anti-ABCC3 nanobody followed by ReadyTag anti-6-His mAb (1:1500; clone 6-HIS, BioXCell) and APC goat anti-mouse IgG antibody (1:1500; clone Poly4053, Biolegend) for 20 min at 4 °C. Cells were analyzed on a Gallios flow cytometer (Beckman Coulter) and results were visualized at Kaluza analysis software (Beckman Coulter). To estimate the equilibrium dissociation constant (K_D), nanobodies were conjugated to fluorescein-5-isothiocyanate using FluoroTag™ FITC Conjugation Kit (Sigma-Aldrich), and total and non-specific binding was analyzed with GraphPad Prism v.6.01.