Neuro2a mouse neuroblastoma cells

Rat MOR cDNA was epitope-tagged with HA at N-terminus and inserted in pcDNA vector with neomycin resistance gene. The construct was transfected into Neuro2A mouse neuroblastoma cells (ATCC) and grown in medium containing 0.5 mg/mL G418 (Geneticin). Clonal cells stably expressing MOR were screened by [³H]diprenorphine binding assay. Two clonal cell lines expressing MOR at 1–2 pmol/mg protein (clones H38 and H16) were used for the internalization experiment (N2A-HA-rMOR). Cells were cultured in 10 cm dishes an incubator maintained at 37 °C with 5% CO₂ in humidified air in MEM (Minimum Essential Medium, ref 41500, Gibco, NY) supplemented with 10% FBS and penicillin, streptomycin, and amphotericin (A5955, Sigma, MO) and grown to 80% confluence.

For mixed glial cultures, postnatal day 0–3 (P0–3) mouse forebrains were dissociated into single cells with trypsin and plated onto poly-L-lysine-coated glass coverslips. The cells were then directly transfected or passaged once and transfected. Oli-neu mouse oligodendrocyte precursor cell line (Jung et al. 1995 (link), a gift from Dr. Jacky Trotter, Mainz) was maintained in DMEM/F12 containing 1% horse serum and N2 supplement (Invitrogen, USA). Neuro-2a mouse neuroblastoma cells (ATCC, USA) were maintained in DMEM containing 10% FBS. Plasmids were transfected into these cells using Lipofectamine 2000 (Life Technologies, USA) according to the manufacturer’s instructions.

[20-³H]Phorbol 12,13-dibutyrate ([³H]PDBu) (17.2 Ci/mmol) was obtained from PerkinElmer Life Sciences. PDBu and phorbol 12-myristate 13-acetate (PMA) were purchased from LC Laboratories (Woburn, MA). Phosphatidyl-L-serine (PS), phosphatidylcholine (PC), and sphingosine were from Avanti Polar Lipids (Alabaster, AL) and the isopropyl O-D-thiogalactopyranoside (IPTG) was from Sigma (St. Louis, MO). LNCaP human prostate cancer cells, Neuro-2a mouse neuroblastoma cells, HEK-293 cells, fetal bovine serum (FBS), RPMI 1640 medium, and L-glutamine were from the American Type Culture Collection (Manassas, VA). Reagents used for culturing bacteria (LB Broth, LB agar plates with ampicillin, ampicillin solution, etc.) were from K-D Medical, Inc. (Columbia, MD). The primers, the High Fidelity Polymerase, and the ligase used for cloning, the chemically competent MaxEfficiency DH5αT1^R and BL-21(DE3) cells, the DMEM without phenol red used for confocal analysis, the Lipofactamine and Plus reagent, Lipofectamine 3000, and the GST spin purification kit were from Thermo Scientific (Pittsburgh, PA). The QIAquick PCR Purification Kit and the QIAprep Spin Miniprep Kit were from Qiagen (Germantown, MD). The pHTN HaloTag(R) CMV-neo vector and the kit for purification of Halo-tagged proteins were from Promega (Madison, WI).