Topro 3 iodide t3605

Human Type I PDZ GPCR cDNAs were purchased from the Missouri S&T cDNA resource center (cdna.org) or cloned from human brain cDNA library (provided by Professor Ning Zheng, HHMI, University of Washington Department of Pharmacology). Human α-syntrophin cDNA was purchased from OriGene Technologies, (Rockville, MD, USA). Human FLAG-Scribble was provided by Professor Jon Huibregtse (Department of Molecular Sciences, University of Texas at Austin). cDNAs were subcloned into pGlue (provided by Professor Randall T. Moon, HHMI, University of Washington Department of Pharmacology) using In-Fusion HD cloning technology (Clontech).
(R)-(−)-phenylephrine hydrochloride (P6126) was purchased from Sigma (St Louis, MO, USA). Anti-FLAG antibodies were purchased from Sigma (2368). Alexa fluor 633 goat-anti Rabbit IgG (A-21070) and Alexa fluor 568 Goat anti-rat (A-11011) antibodies from Life Technologies. Topro-3 iodide (T3605) is from Life Technologies. Rabbit polyclonal α-syntrophin (H-65, sc-50460), and rabbit polyclonal Scrib (H-300, sc-28737) antibodies from Santa Cruz Biotechnology. Mouse monoclonal Anti-syntrophin (1351, ab11425) antibody and rabbit polyclonal anti-Myc tag antibody (ab9106) from Abcam (Cambridge, MA, USA). Anti-HA mouse mAb (6E2, #2367) from Cell Signaling (Danvers, MA, USA). IRdye 680 goat antimouse IgG and IRdye 800cw goat antirabbit IgG from Li-Cor (Lincoln, NE, USA).

A5-FITC (556419), A5-V450 (560506), A5-APC (550475), 10x A5 binding buffer (556454), anti-mouse Ly6G APC-Cy7 (560600), anti-mouse SiglecF PE (552126), anti-human CD11b PE (555388), mouse FcR blocking reagent (553141) and anti-human CD45 PE-Cy7 (557748) were purchased from BD Biosciences. TO-PRO-3 iodide (T3605) and PO-PRO-1 (P3581) were purchased from Life Technologies. The following antibodies were purchased from eBioscience; anti-mouse CD4 PE-Cy7 (25-0042-82), anti-mouse CD8a PE (12-0081-82), anti-human CD3 APC-Cy7 (17-0031-83) and anti-human CD56 Pe-Cy5 (15-0567-42). Anti-mouse CD45.2 FITC (109806) and anti-mouse CD64 PE-Cy7 (139313) were purchased from BioLegend. Anti-human CD14 FITC (130-098-063) and human FcR blocking reagent (130-059-901) were purchased from MAC Miltenyi Biotech. Ficoll Paque Premium was purchased from GE Healthcare Life Science (17-5442-02). Complete media was prepared using RPMI 1640 medium (Life Technologies, 22400-089), 50 IU ml⁻¹ penicillin and 50 μg ml⁻¹ streptomycin mixture (Life Technologies, 15140122), 10% (vol/vol) fetal calf serum (FSC) (Gibco, 10099-141) and 0.2% (vol/vol) MycoZap (Lonza, VZA-2012). FACS buffer was prepared using 10% FCS, 2x A5 binding buffer and 2 mM EDTA (Sigma, 1001710526) in PBS.