Apc goat anti mouse igg antibody

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.

Lipopolysaccharides (LPS) and BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488 was supplied by the Beyotime. NBT/BCIP tablets were supplied by Roche Diagnostics GmbH. Alizarin Red, Fluorescein diacetate (FDA) and Propidium Iodide (PI) were obtained from Solarbio Life Sciences. RNA‐Quick Purification Kit was obtained from ES Science. HiScript® II Q RT SuperMix for qPCR and AceQ® qPCR SYBR Green Master Mix were provided by Vazyme. CD68 monoclonal antibody (ED1), CD80 (B7‐1) monoclonal antibody (3H5) and Goat anti‐rabbit IgG (H+L) secondary antibody, PE‐Cyanine 5.5 were supplied by Invitrogen. CD206 antibody was obtained from Santa Cruz Biotechnology, Recombinant Anti‐CD163 antibody was supplied by Abcam. RUNX2 Rabbit mAb was supplied by Cell Signaling Technology Inc. PE‐anti rat CD86 antibody, APC goat anti‐mouse IgG antibody and APC/Cyanine 7 streptavidin were obtained from Biolegend.