Primary midbrain dopaminergic neurons were derived from ventral mesencephalon of postnatal day 0 mouse pups based on Florence Gaven’s protocol34 . In brief, dissociated cells were seeded at cell culture dishes coated with poly-d -lysinecoated. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air in DMEM/F12 medium containing 10% fetal bovine serum, 50 U/ml penicillin, and 50 g/ml streptomycin. Next day, culture medium was changed to B-27 Plus Neuronal Culture System (Thermo Fisher Scientific, A3653401) containing 50 U/ml penicillin, and 50 g/ml streptomycin. At DIV 7, DA neurons were detected by anti-TH antibody with immunofluorescence (fig. S2B ). Seven-day-old cultures were used for treatment. Adenovirus that expression either GFP, α-synuclein A53T were used to infect Primary midbrain dopaminergic neuron at 7 DIV.
B 27 plus neuronal culture system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The B-27 Plus Neuronal Culture System is a cell culture supplement designed to support the growth and differentiation of neuronal cells. It provides a defined, serum-free formulation optimized for the maintenance and maturation of neuronal cultures.
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Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (3 mentions)
Calcein am, by Beyotime (3 mentions)
Nbactiv4, by Transnetyx (2 mentions)
4d nucleofactor, by Lonza (2 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic solution, by Merck Group (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
B6.129p2 cxcr3tm1dgen j, by Jackson ImmunoResearch (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Neurobasal medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Merck Group (1 mentions)
13 protocols using b 27 plus neuronal culture system
1
Postnatal Midbrain Dopaminergic Neuron Culture
2
Culturing Mouse Neuroblastoma and Cortical Neurons
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Mouse neuroblastoma x rat neuron hybrid ND7/23 cells (ECACC 92090903, Sigma Aldrich) were grown in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM; Thermo Fisher Scientific, cat. no. 41965062) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, cat. no. 10270106), 1% penicillin-streptomycin (Sigma Aldrich, cat. no. P0781), 1% sodium pyruvate (Thermo Fisher Scientific, cat. no. 11360039) and 1% L-glutamine (Thermo Fisher Scientific, cat. no. 25030024) at 37 °C, 5% CO2. FBS was heat-inactivated by incubation at 56 °C for 30 minutes (min). For more details, see Supplementary Information.
Primary mouse cortical neurons (MCN) from C57BL/6 embryonic day-17 mice were obtained from Thermo Fisher Scientific (cat. no. A15586). Neurons were thawed following the manufacturer’s recommendations and seeded in eight-well Lab-Tek II chambered #1.5 German coverglass (Thermo Fisher Scientific, cat. no. 155409). Neurons were maintained in B-27™ Plus Neuronal Culture System (Thermo Fisher Scientific, cat. no. A3653401), supplemented with 1% penicillin-streptomycin (Sigma Aldrich, cat. no. P0781). Half of the culturing medium was changed twice per week.
Primary mouse cortical neurons (MCN) from C57BL/6 embryonic day-17 mice were obtained from Thermo Fisher Scientific (cat. no. A15586). Neurons were thawed following the manufacturer’s recommendations and seeded in eight-well Lab-Tek II chambered #1.5 German coverglass (Thermo Fisher Scientific, cat. no. 155409). Neurons were maintained in B-27™ Plus Neuronal Culture System (Thermo Fisher Scientific, cat. no. A3653401), supplemented with 1% penicillin-streptomycin (Sigma Aldrich, cat. no. P0781). Half of the culturing medium was changed twice per week.
3
Primary Cortical Neuron Isolation and Culture
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Primary embryonic cortical neurons from pregnant rats (embryonic day 19) were prepared as previously described [38 (link)]. Briefly, the cortex of the brain was dissected from the embryo and purified by a papain dissection kit (Worthington Biochemical, Lakewood, NJ, USA). Twenty-four-well plates were coated with 0.1 mg/mL poly-D-lysine (PDL) for 3 h and washed with MilliQ water. Cortical neurons were plated on the PDL-coated plates at a density of 200,000 cells per well in 1 mL of B-27 Plus Neuronal Culture System (Thermo Fisher Scientific; #A3653401) containing 2 mM GlutaMAX (Thermo Fisher Scientific) and 1% antibiotic antimycotic solution (Sigma-Aldrich, St. Louis, MO, USA). DIV 14-30 neurons were used for the experiments.
4
Primary Mouse Cortical Neuron Culture
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Primary mouse cortical neuronal cultures were obtained from E16 ICR mouse embryos (Laboratory Animal Facility, Hong Kong University of Science and Technology). Briefly, cerebral cortices were removed from E16 ICR mouse embryos and mechanically dissociated in PBS with glucose (18 mM). Neurons were then seeded onto poly-L-lysine (1 mg/mL) coated 96-well plates at 5 × 104 cells/well in B27 Plus Neuronal culture system (Thermo Fisher Scientific), supplemented with 2 mM glutamine, 5 μM mercaptoethanol, 100 U/mL penicillin and 100 μg/mL streptomycin. The cultured neurons were maintained in a humidified incubator at 37 ℃ with 5% CO2 for 7 days prior to use.
5
Cultured Rat Neuron Manipulation
6
Cultured Rat Neuron Manipulation
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Hippocampi and midbrains were dissected from P0 Sprague-Dawley rat pups of both sexes. Rat maintenance and care followed policies advocated by NRC and PHS publications and approved by the Institutional Animal Care and Use Committee (IACUC), Janelia Research Campus. Tissues were digested with papain and gently triturated and filtered through a 40 μm filter. Neurons were electroporated (Lonza 4D-nucleofactor) with Tet-On Arl13b-RhoA (hippocampal neurons) or Tph2-Cre (tryptophan hydroxylase 2-Cre, midbrain neurons) and plated in poly-D-lysine coated dishes and cultured in NbActiv4 (BrainBits) at 37°C and 5% CO2. A week after plating, the neuronal cultures were fed with B-27 plus neuronal culture system (Thermo Fisher Scientific). AAV transduction (Syn1-GRAB-HTR6-cilia, FLEX-on hM3DGq-DREADD, FLEX-on farnesylated SNAP, or FLEX-on tdTomato) were applied at DIV10. 100 ng/ml doxycycline was added at DIV 6 or DIV14 and removed the next day for Arl13b-RhoA experiments. Images were collected between DIV 9 and DIV 28 (Alr13b-RhoA, hippocampal culture only) or DIV21-35 (hippocampal and midbrain co-culture). JF552-STL (SNAP-labeled neurons) was applied the day prior to imaging (100 nM, 2 h).
7
Isolation and Culture of Cortical and Hippocampal Neurons
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Cortical and hippocampal neurons from P0 to P1 WT or KO mouse pups (B6.129P2-Cxcr3tm1Dgen/J; the Jackson Laboratory, strain #005796) were obtained as described previously (118 (link)), with minor modifications. Briefly, papain-dissociated cells were filtered through a 0.4-μm cell strainer (Corning, #431750) to enrich for neurons, centrifuged at 500g for 5 min to eliminate small debris, and suspended in complete primary neuronal medium consisting of B-27 Plus Neuronal Culture system (Thermo Fisher Scientific, #A3653401) and 1× GlutaMAX (Thermo Fisher Scientific, #35050061) without antibiotics. Cells were seeded at 50,000 to 150,000 live cells/cm2 into plates coated with poly-d -lysine (0.01%, w/v; Sigma-Aldrich, P6407). Medium was fully exchanged 1 day after plating (DIV 1) with subsequent half-medium exchanges every 3 to 4 days.
8
Nanoparticle Uptake in Primary Rat Cortex Neurons
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Primary rat cortex neurons were plated
on Corning BioCoat collagen IV 100 mm TC-treated culture dishes in
a 5% CO2 atmosphere at 37 °C, using the Gibco B-27
Plus Neuronal culture system as medium for 30 days to allow the axons
to fully grow. 2.1 × 109 particles per milliliter
of NNTs were suspended in medium and incubated with cells for 20 min.
The cells were then washed to remove free NNTs with DPBS. The neurons
were incubated with 75 nM LysoTracker Red DND-99 (Life Technologies)
for 30 min at 37 °C and washed 3 times with DPBS. The neurons
were then incubated with 0.5 mL of Invitrogen NucBlue Live ReadyProbes
reagent (Hoescht) for 30 min at 37 °C and washed 3 times with
DPBS.
on Corning BioCoat collagen IV 100 mm TC-treated culture dishes in
a 5% CO2 atmosphere at 37 °C, using the Gibco B-27
Plus Neuronal culture system as medium for 30 days to allow the axons
to fully grow. 2.1 × 109 particles per milliliter
of NNTs were suspended in medium and incubated with cells for 20 min.
The cells were then washed to remove free NNTs with DPBS. The neurons
were incubated with 75 nM LysoTracker Red DND-99 (Life Technologies)
for 30 min at 37 °C and washed 3 times with DPBS. The neurons
were then incubated with 0.5 mL of Invitrogen NucBlue Live ReadyProbes
reagent (Hoescht) for 30 min at 37 °C and washed 3 times with
DPBS.
9
Primary Cell Culture in Tyrode's Buffer
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The composition of Tyrode’s buffer was 140 mM NaCl, 4 mM KCl, 1.8 mM CaCl2, 5 mM HEPES, 0.33 mM NaH2PO4, 0.1 mM MgCl2 and 5.5 mM glucose. HEPES was purchased from Dōjindo Laboratories; NaCl, KCl, CaCl2, NaH2PO4, MgCl2 and glucose from FUJIFILM Wako Pure Chemical Corporation. B-27™ Plus Neuronal Culture System (#A3653401, Gibco) containing penicillin-streptomycin solution (#P4333, Sigma-Aldrich) was used to culture primary cells, and Neurobasal medium (#21103–049, Gibco) containing B27 supplement (#17504044, Gibco), GlutaMAX (#35050061, Gibco) and penicillin-streptomycin solution was used to harvest the cells.
10
Cryopreserved Mouse Hippocampal Neuron Culture
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Cryopreserved primary mouse hippocampal neurons were commercially obtained from Gibco-Life Technologies (Carlsbad, CA, USA) and cultured in B-27 Plus Neuronal Culture System (Gibco-Life Technologies, Carlsbad, CA, USA). After thawed and seeded in tissue culture flasks containing 5 mL media supplemented with neuronal growth supplement, 1% penicillin/streptomycin and 5% fetal bovine serum, neurons were kept in a humidified incubator filled with 5% CO2 and 95% air at 37°C. The culture media were replaced every other day, until neurons reached about 70% confluency and were ready for experiments without subculturing.
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