Hek293 cells

Rat astrocytic C6 glioma cells and human embryonic kidney (HEK)-293 cells were purchased from RIKEN Cell Bank (Saitama, Japan). Mouse embryonal carcinoma P19 cells were obtained from ATCC (Manassas, VA, USA). Mouse microglial BV-2 cells are a generous gift from Dr. Eui-Ju Choi (Korea University, Seoul, Korea) [28 (link)]. Poly-L-lysine, all-trans retinoic acid (ATRA), Hoechst33342, propidium iodide (PI), A23187, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), 2-mercaptopyridine N-oxide sodium (pyrithione) and N,N,N’,N’-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) were purchased from Sigma-Aldrich fine Chemicals (St. Louis, MO, USA). Acetoxymethyl esters of Fluo-3, Rhod-2 and FluoZin-3 were provided by Molecular Probes (Eugene, OR, USA). Both EGTA and BAPTA-AM were supplied by Dojindo (Kumamoto, Japan). Dulbecco’s Modified Eagle Medium (DMEM) and alpha minimal essential medium (αMEM) were provided by Wako (Osaka, Japan). EDTA was purchased from Nacalai Tesque (Kyoto, Japan). Other chemicals used were all of the highest purity commercially available.

BWZ.36 cells were provided by Dr. N. Shastri (University of California Berkeley, Berkeley, CA, USA) and maintained in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal calf serum (FCS, Gibco, Tokyo, Japan), 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine, and 50 μM 2-mercaptoethanol under 5% CO₂ at 37°C. The retrovirus-packaging cell line, Plat-E cells, were provided by Dr. T. Kitamura (The University of Tokyo, Tokyo, Japan) and maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) supplemented with 10% heat-inactivated FCS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine, 50 μM 2-mercaptoethanol, and 20 mM HEPES (D10 medium). HEK293 cells (Riken cell bank, Ibaraki, Japan) were maintained in D10 medium. Experiments using human cell lines and genetic recombination experiments were conducted in accordance with a comprehensive, high quality care program, which was approved by the Life Science Research Committee of the Graduate School of Frontier Sciences at The University of Tokyo and the Life Science Committee of The University of Tokyo.

Human embryonic kidney (HEK) 293 cells (RIKEN Cell Bank, Tsukuba, Ibaraki, Japan) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MI, USA). Cell cultures were incubated at 37 °C in a 5% CO₂ humidified incubator. Cells were cultured in culture plates to 80–90% confluence, and transiently transfected with cDNA plasmids using Lipofectamine 2000 transfect reagent (Thermo Fisher Scientific, Waltham, MA, USA) diluted in Opti-MEM reduced serum medium (Thermo Fisher Scientific, Waltham, MA, USA) according to the reagent instructions.