Pregnant rats were anesthetized with CO2/O2, euthanized before taking the E18 embryos out from their uteri. Embryos were then decapitated, skulls were opened, brains were collected in petri dishes with HBSS on ice. Hemispheres were separated, meninges were carefully stripped away, and hippocampi were dissected on ice and triturated in 1xHBSS (Invitrogen) after digestion by papain and DNase (Worthington for 10 min at 37°C). Transfections were performed using the Amaxa nucleofector system following the manufacturer’s manual. The final concentration for the EB3-tdTomato or Farnesylated-GFP plasmid was 1 μg, for tdTomato-TACC3 we used 0.3 or 0.5 μg. Empty pcDNA 3.1 was used to make up to 3 μg of DNA for 5 3 106 cells per each transfection mix as per the manufacturer recommendation. After electroporation, neurons were plated on poly-L-lysine coated coverslips or tissue culture chambers (Sarstedt, for live-imaging) in Neurobasal/B27 medium (Invitrogen) and were maintained in culture for 24 to 72h or 7- days at 37°C with 5% CO2 before use.
Neurobasal b27 medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Neurobasal/B27 medium is a cell culture medium specifically formulated for the growth and maintenance of primary neuronal cells. It provides essential nutrients and growth factors to support the survival and differentiation of neurons in vitro. The medium is a combination of the Neurobasal base medium and the B27 supplement, which together create an optimized environment for neuronal cell culture.
Automatically generated - may contain errors
Lab products found in correlation
1xhbss, by Thermo Fisher Scientific (6 mentions)
Papain, by Worthington (5 mentions)
Tissue culture chambers, by Sarstedt (4 mentions)
Poly d lysine, by Merck Group (4 mentions)
Dnase, by Worthington (3 mentions)
Poly l lysine (pll), by Merck Group (3 mentions)
Penicillin streptomycin, by Corning (2 mentions)
Dnase, by Merck Group (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Ovomucoid, by Worthington (2 mentions)
Permanox chamber slides, by Merck Group (2 mentions)
Clone ap20, by Merck Group (2 mentions)
Aav1 hsyn turborfp, by Merck Group (2 mentions)
Laminin, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Glutamate, by Merck Group (2 mentions)
2 mercaptoethanol, by Merck Group (2 mentions)
Bsa solution, by Thermo Fisher Scientific (2 mentions)
36 protocols using neurobasal b27 medium
1
Embryonic Rat Hippocampal Neuron Isolation
2
Microglial Modulation of Neuronal Integrity
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Cortices from P1 rats were finely minced and digested for 30 min at 37 °C in DPBS (Gibco) containing papain (Worthington Biochemicals) and DNase (Sigma-Aldrich). papain was inhibited by the addition of ovomucoid (Worthington Biochemicals). Neurons were plated at a density of 60,000 cells/cm2 on poly-D-lysine pre-coated 8-well Permanox chamber slides (Sigma-Aldrich) in Neurobasal/B27 medium (Invitrogen) and cultured for 10 days. Rat BMDMs were cultured in RPMI-1640 with 10% heat-inactivated FBS (Invitrogen), 1% penicillin-streptomycin (Corning), and 10 ng/ml rat M-CSF (#400–28, Peprotec). BMDMs were plated on 25 μg/ml fibrin-coated plates with 20 μg/ml 5B8 or IgG2b for 24 h and were lifted with PBS-EDTA as described16 (link) and added to cortical neuron cultures for two days, fixed with 4% PFA and immunostained with anti-MAP-2 (1:1000; clone AP20, EMD Millipore) and thresholded images were quantified with the NeurphologyJ plug-in in ImageJ. 2.5 × 1010 GC/ml of AAV1.hSyn.TurboRFP (University of Pennsylvania Vector Core) was used to transduce primary cortical neurons for 8 d prior to the addition of fibrin-stimulated BMDMs for 12 h. RFP images were thresholded and the neurite fragments were analyzed using the ImageJ plugin ‘Analyze Particles’. Quantification was performed by an observer blinded to the experimental treatments.
3
Isolation and Culture of Rat Hippocampal Neurons
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Primary hippocampal neurons were obtained from fetal SD rats as described previously (Kaech & Banker, 2006 ). In brief, hippocampal tissues were dissociated by mild mechanical trituration, plated at 4 × 105 cells·ml−1 in 24‐well culture plates previously coated with 1 mg·ml−1 poly‐d ‐lysine (Cat No. P6407; Sigma), and maintained in DMEM supplemented with 5% FBS (Cat No. SH30048.03; Hyclone). Three days after plating, the medium was replaced with Neurobasal/B27 medium (Cat No. 21103049; Invitrogen) containing cytosine arabinoside (final concentration 5 μM; Cat No. C6645; Sigma) to halt the proliferation of non‐neuronal cells. One third of the medium was replaced with fresh medium every 3 days. The cultures were maintained at 37°C in a humidified 5% CO2 atmosphere. To determine the effects of BDNF‐treated astrocyte‐CM on hippocampal neuronal survival after thrombin treatment, hippocampal neuronal cultures were exposed to 50 U·ml−1 thrombin alone or in combination with BDNF‐treated astrocyte‐CM or anti‐CNTFRα neutralizing antibody for 48 hr.
4
Purification of Primary Retinal Ganglion Cells
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Primary retinal ganglion cells (RGCs) were isolated according to the two-step immunopanning method as previously reported[11 (link)]. After the deep anesthesia by isoflurane, the animals were sacrificed by CO2 asphyxiation followed by decapitation. Then the whole retina was isolated and incubated in a papain solution (16.5U/mL, Sigma-Aldrich, US) for 30 min. Next, macrophages and endothelial cells were removed from the cell suspension by panning with an antimacrophage antiserum (Accurate Chemical, Westbury, NY). RGCs were specifically bound to the panning plates containing anti-Thy1.1 antibody (2 μg/ml; Chemicon, US) and released by trypsin treatment. RGCs were grown in serum-free basal medium (Neurobasal/B27 medium; Invitrogen, US).
5
Culturing and Analyzing Primary Cortical Neurons
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Briefly, cortical tissues were dissected from the pups of different dams within 24 h after birth, digested with trypsin, and mechanically triturated in Neurobasal/B27 medium (Invitrogen, USA). Then, neurons were centrifuged, re-suspended in Neurobasal/B27 medium, and cultured in an incubator. Half of the Neurobasal medium was changed every three days. The detailed processes were described in our previous study [62 (link)]. Axonal morphology was observed on DIV2 and DIV5 using an inverted microscope (IX73, Olympus, Japan) and measured using Image J.
6
FKBP-Mediated Regulation of Eph Clustering
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COS-7 cells used for homo-FRET experiments, immunostainings, and blue native PAGE were transfected using FUGENE6 (Roche). HeLa cells were transfected using a Calcium-Phosphate transfection kit (Invitrogen) according to the manufacturer’s protocol. 12–16 h before stimulation, cells were starved in growth medium containing dialyzed 0.5% fetal bovine serum (HyClone). For FKBP domain–containing constructs FK506 (300 nM) was added to the growth medium after transfection to reduce Eph clustering.
Primary hippocampal neurons were dissected from embryonic day 18.5 rat embryos, plated onto glass coverslips (Marienfeld) coated with 1 mg/ml poly-d -lysine (Sigma-Aldrich) and 5 µg/ml laminin (Invitrogen), and cultured in Neurobasal-B27 medium (Invitrogen; Lauterbach and Klein, 2006 (link)). Neurons were transfected using Amaxa nucleofection kit (Lonza).
Primary hippocampal neurons were dissected from embryonic day 18.5 rat embryos, plated onto glass coverslips (Marienfeld) coated with 1 mg/ml poly-
7
Microglial Modulation of Neuronal Integrity
8
Embryonic Rat Hippocampal Neuron Isolation
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Pregnant rats were anesthetized with CO2/O2, euthanized before taking the E18 embryos out from their uteri. Embryos were then decapitated, skulls were opened, brains were collected in petri dishes with HBSS on ice. Hemispheres were separated, meninges were carefully stripped away, and hippocampi were dissected on ice and triturated in 1xHBSS (Invitrogen) after digestion by papain and DNase (Worthington for 10 min at 37°C). Transfections were performed using the Amaxa nucleofector system following the manufacturer’s manual. The final concentration for the EB3-tdTomato or Farnesylated-GFP plasmid was 1 μg, for tdTomato-TACC3 we used 0.3 or 0.5 μg. Empty pcDNA 3.1 was used to make up to 3 μg of DNA for 5 3 106 cells per each transfection mix as per the manufacturer recommendation. After electroporation, neurons were plated on poly-L-lysine coated coverslips or tissue culture chambers (Sarstedt, for live-imaging) in Neurobasal/B27 medium (Invitrogen) and were maintained in culture for 24 to 72h or 7- days at 37°C with 5% CO2 before use.
9
Isolation of Embryonic Mouse Hippocampal Neurons
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Hippocampal neurons were obtained from E18 mouse embryos as previously described [14 (link)]. Briefly, embryonic hippocampi were isolated in Hank’s balanced salt solution (HBSS) with 10 mM HEPES, pH 7.4 and 0.25% trypsin and incubated for 15 minutes at 37°C. Trypsin was eliminated by washing three times with HBSS, and tissue was homogenized by pipetting. Homogenized neurons were plated onto 1mg/ml poly-lysine and 5 μg/ml laminin-coated 15 mm cover slips containing plating medium (MEM, 20% Glucose and 10% horse serum, Invitrogen) and incubated for 1.5 hours at 37°C and 5% CO2. After that, the medium was changed to Neurobasal/B27 medium (Invitrogen).
10
Neuron Transfection and Isolation
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Neurons were transfected by in utero electroporation at E15 and transfected cortices were isolated (as explained above) two days later. Isolated cortices were triturated in 1xHBSS (Invitrogen) containing papain and DNase at 37 °C (Worthington). Neurons were plated on poly-L-lysine coated glass coverslips in Neurobasal/B27 medium (Invitrogen), maintained in culture for 24 hours for time-lapse imaging.
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