Yasumoto et al
Genetic manipulation of primate neuronal cultures
Supplementary information: methods
Animals: Fetuses of cynomolgus monkeyat 80 days of gestation (E80) were purchased from ShinNippon Biomedical Laboratories (Kagoshima, Japan). Allfetuses were obtained by Cesarean section and immediatelykilled by decapitation. This study was approved by the AnimalCare and Use Committee of the Graduate School ofAgricultural and Life Sciences, the University of Tokyo.
Preparation of fetal cerebral cortex and cryopreservation: Fetal brains were removed under sterile condition in ice-cold isolationmediumconsisting of equal volumes of Ca2+- andMg2+-free phosphate-buffered saline and Dulbecco’sModified Eagle’s Medium (DMEM) (Sigma, St. Louis, MO) with 25 g/ml streptomycin and 50 units/ml penicillin. After the meninges were removed, thecerebral cortex was mechanically cut intosmall pieces. For cryopreservation, tissues were placed in aliquots incryoprotective medium [10% fetal bovine serum(FBS) and 10%dimethyl sulphoxide (DMSO) (Sigma) in DMEM], then first frozen in the Cryo 1 CFreezing Container (Nalgene, Japan) to make the temperature dropped at 1C per min until it reached -80C; aliquots were then transferred to a liquid nitrogen tank for long-term storage.
Preparation of primary cultures: Cryopreserved cortical tissues were thawed rapidlyin a water bath at 37 C, rinsed in the IM media to remove DMSO, and then treated withdigestion buffer [10 U/mlpapain,0.1 mg/ml DNase I, 0.2 mg/mlcysteine, 0.2 mg/ml albumin, and 5 mg/ml glucose (Sigma)] at 37Cfor 30 min. The cells were dissociated by trituration with a pipette and centrifugedthree times at 800 rpm for5 min. The cells were suspended inserum-containing medium (DMEM supplemented with 10 % FBS), andwere then plated onto culture dishes coated with 0.5%polyethylenimine (PEI) at a density of 1104 cells/mm2 forhigh-density culture or 1103 cells/mm2 for low-density culture. All cultures weremaintained at 37 C in 95% humidified air and 5% CO2.
Immunofluorescent cell staining: We followed the published protocol. Primary antibodies used in this study are as follows: anti-MAP2 polyclonal antibody (Chemicon, Temecula, CA; 1:1000 dilution), anti-MAP2 monoclonal antibody (Roche Diagnostics, Basel, Switzerland; 1:2000 dilution),anti-GFAP (DAKO, Tokyo, Japan; 1:1000), anti-Nestin (Chemicon; 1:200), anti-DCX (Chemicon;1:1000), anti-CaMKII (Zymed, San Francisco, CA; 1:200), and anti-GAD65 (Santa Cruz Biotechnology, Santa Cruz, CA; 1:20). Alexa 488- or 568-conjugated secondary antibodies are from Invitrogen (Eugene, OR;1:400). TO-PRO-3 (Invitrogen)was used for nuclear staining. Images randomly selected from 50 fields in 4 independent culture preparations were used for quantitative measurement.
Plasmid transfection: A GFP expression construct was transfected into the culture by using LipofectAMINE2000 (Invitrogen), according to the protocol from the vendor. Twenty-four hours later at 2 or 13DIV, transfection efficiency was examined.
Statistics: All statistical analyses were performed using one-way ANOVA, followed by the Dunnett’s multiple comparisons test.
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Supplementary figure and table legends
Figure
a.Immunostaining of neuronal and glial markers. Neurons ranging from 1.5 to 14 DIV were stained with Ab to MAP2(red), GFAP (Green), and TO-PRO-3 nuclear staining (blue). The mean s.e.m (n50/group) of number of MAP2-, or GFAP- positive cells are shown. Scale bars, 50m.*P 0.05.
b. Immunostaining of neuronal progenitor and premature neuron markers. Nestin-positive cells (neuronal progenitors) are dramatically reduced during culture. Relative ratio of doublecortin (DCX)-positive cells (premature neurons) to MAP2-positive mature neurons was decreased during culture. Green, Nestin or DCX; red, MAP2; blue, nucleus.
Table
Influence of cell density on transfection efficiency. Number of cells seeded and that of viable cells at the time of transfection are shown. The mean±s.e.m (n=50/group) of transfection efficiency (TE) at various cell densities was counted.
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