After collection, explants were immediately placed in F12 media (Gibco, Waltham, MA) containing collagenase (1.25 mg/ml) (Sigma-Aldrich, St. Louis, MO) and incubated (37 °C and 5% CO2) for 45 min. The collagenase step was repeated for another 45 min. Afterward, explants were incubated in F12 media containing trypsin (0.025%, Sigma-Aldrich, St. Louis, MO) mixture for 30 min followed by F12 media-containing FBS (Sigma-Aldrich, St. Louis, MO) for 15 min. Thereafter, explants were washed three times with F12 media and mechanically dissociated with a glass pipette until the solution turned cloudy indicating a successful dissociation. Dissociated cells were filtered through a 0.22-μm filter (BD Falcon, Franklin Lakes, NJ) and centrifuged (2400 rpm) for 2 min. After removing the supernatant, the cells were resuspended in neurobasal media (Sigma-Aldrich, St. Louis, MO) containing B-27 supplement (Life Technologies, Carlsbad, CA), PSN antibiotics (Gibco, Waltham, MA), 0.5 mM l -glutamine (Sigma-Aldrich, St. Louis, MO) and NGF (5 ng/ml, Alomone Labs, Jerusalem, Israel). The resuspended cells were plated on laminin-coated cover slides (Neuvitro, Vancouver, WA).
Neurobasal media
Manufactured by Merck Group
Neurobasal media is a cell culture medium designed for the growth and maintenance of neuronal cells. It provides a defined, serum-free environment that supports the survival and differentiation of neurons. The core function of Neurobasal media is to facilitate the in vitro cultivation of neuronal cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Neurobasal media, by Thermo Fisher Scientific (4 mentions)
Trypsin, by Merck Group (3 mentions)
L glutamine, by Merck Group (3 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
Dnase, by Merck Group (2 mentions)
Uridine, by Merck Group (2 mentions)
Collagenase, by Merck Group (1 mentions)
F12 media, by Thermo Fisher Scientific (1 mentions)
F12 media, by Merck Group (1 mentions)
0.22 μm filter, by BD (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Nerve growth factor (ngf), by Alomone (1 mentions)
Potassium chloride (kcl), by Merck Group (1 mentions)
Dnase 1, by Roche (1 mentions)
Exoquick tc solution, by System Biosciences (1 mentions)
Nerve growth factor (ngf), by Merck Group (1 mentions)
Lab tek 2 4 well chambered cover glass, by Thermo Fisher Scientific (1 mentions)
5 fluro 2 deoxyuridine, by Merck Group (1 mentions)
8 protocols using neurobasal media
1
Isolation and Culture of Primary Sensory Neurons
2
Rat Cortical Neuron Isolation and Transduction
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The cortex from the brains of E17.5 rat pups were harvested and stored in Hank’s Balanced Salt Solution (without calcium, without magnesium) (HBSS -/-) (Sigma Aldrich). The tissue was washed with HBSS (-/-) and then incubated with 0.0035% Trypsin (Sigma Aldrich) for 15 minutes. DNAse (10µg/mL)(Sigma Aldrich) was then added at a ratio of 1:1 (v/v), and the tissue was re-suspended in 1mL triturating solution (1% Albumax, 25mg Trypsin Inhibitor, 10µg/mL DNAse) (Sigma Aldrich). Neurobasal media (ThermoFisher), supplemented with 2 mM L-Glutamine (Sigma Aldrich), 1% Penicillin/Streptomycin (Sigma Aldrich) and 1 x B-27 (Sigma Aldrich) was then added at a ratio of 1:5 (Triturating solution:Neurobasal media). Cells were then plated onto Poly-D-Lysiene coated coverslips, in the wells of a 24 well-plate at a density of 9.365 x 104 cells/cm2. 1.5 x 105 viral genomes (vg) per cell of AAV9 was added to cultures after 5 days in vitro (DIV). Half of the culture media was replaced with fresh media every 3 days. 7-days post-transduction (13 DIV), cells were treated with CPT (10μM) where indicated, and fixed using 4% paraformaldehyde or methanol:acetone (50:50).
3
Cerebellar Neuroinflammation Induction and Analysis
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Neuronal/glial cerebellar cultures were derived from mice using an established protocol [36 (link), 71 (link)]. Isoflurane anesthetized mice were killed by decapitation at post-natal day 4 (P4). The cerebella were dissected away from the meninges and choroid plexus. Minced cerebellar tissue was trypsinized for 15 min at 37 °C and then triturated in Hank’s balanced salt solution containing 10 U/mL DNAse I (Roche Diagnostics). The cells were centrifuged at 2000 rpm for 7 min and resuspended in Neurobasal media (Sigma) containing 4 mM glutamine, 10% FBS, 100 U/mL penicillin/streptomycin, and 25 mM KCl (Sigma-Aldrich). After counting, 7.5 × 105 cells were plated on precoated poly-d -lysine glass coverslips in 24-well plates. Cultures were maintained at 37 °C, 5% CO2, and media were changed every 2 days. On day 6 in culture, the cells were treated with LPS (Sigma #L2630) at 100 ng/mL concentration for 3 h. They were then fixed in 4% paraformaldehyde for immunohistochemical staining.
In vivo LPS treatment in wild-type mice was performed as previously described [36 (link)]. Briefly, LPS in PBS was administered intraperitoneally at a dose of 750 μg/kg for seven consecutive days. The control mice received the vehicle PBS alone. After 7 days of injections, mice were killed for immunohistochemical analysis.
In vivo LPS treatment in wild-type mice was performed as previously described [36 (link)]. Briefly, LPS in PBS was administered intraperitoneally at a dose of 750 μg/kg for seven consecutive days. The control mice received the vehicle PBS alone. After 7 days of injections, mice were killed for immunohistochemical analysis.
4
Isolation and Characterization of Extracellular Vesicles
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Extracellular vesicles (EVs) were purified from conditioned media [CM: 24 h, serum-free neurobasal media (Sigma)] following a described protocol (Chakrabortty et al., 2015 (link)). Briefly, CM was collected and centrifuged at 2000 × g for 10 min, then passed through a 0.22 µm syringe filter. EVs were precipitated from 10 ml CM by addition of 2 ml ExoQuick TC solution (System Biosciences). Samples were incubated overnight at 4°C. EVs were pelleted by centrifugation at 1500 × g for 30 min at 4°C, the supernatant was discarded. EVs were resuspended in 110 µl PBS with a 10 µl sample used for NanoSight NTA particle analysis and 100 µl used for RNA extraction. Expression of exosomal markers was confirmed by western blotting, where EVs from 10 ml CM were resuspended in 25 µl RIPA buffer, and analysed as described. Antibodies used were Rabbit anti-Alix (Bethyl Laboratories, A302-938A, RRID: AB_10681518) and Rabbit anti-Flotillin 1 (Abcam, ab RRID: AB_941621).
5
Rat Cortical Neuron Isolation and Transduction
6
Embryonic Mouse DRG Neuron Culture
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This study was carried out in accordance with the recommendations of PHS Policy on Humane Care and Use of Laboratory Animals, the Guide for the Care and Use of Laboratory Animals, and the policies and procedures of the University of Central Arkansas. The protocol was approved by the UCA Animal Care and Use committee.
Mouse DRG were collected from embryonic day 12.5 (E12.5) CD1 mice embryos. The DRGs from 5 embryos (~200 total DRGs) of CD1 mice were dissociated and resuspended in 50 μl of Neurobasal media (Invitrogen) containing 2% B27 (Invitrogen) and 50 ng/ml NGF (Harlan Bioproducts) per dissected embryo. Suspended DRG neurons were placed as a drop (2 μl) near one end of each well in Lab TekII 4-well chambered cover glass (Nalge Nunc International), which had been previously coated with poly-D-lysine (0.1 mg/ml) and laminin (2–5 ug/ml). The chambered cover glass was then incubated at 37°C at 5% CO2 for 15 min before 500 μl of Neurobasal media containing 2% B27, 50 ng/ml NGF, 1 μm 5-fluro-2′-deoxyuridine (Sigma), and 1μm uridine (Sigma) was added to each well.
Vincristine (0.4 μM) was added to 14 days in vitro (DIV) cultures to induce axon degeneration.
Mouse DRG were collected from embryonic day 12.5 (E12.5) CD1 mice embryos. The DRGs from 5 embryos (~200 total DRGs) of CD1 mice were dissociated and resuspended in 50 μl of Neurobasal media (Invitrogen) containing 2% B27 (Invitrogen) and 50 ng/ml NGF (Harlan Bioproducts) per dissected embryo. Suspended DRG neurons were placed as a drop (2 μl) near one end of each well in Lab TekII 4-well chambered cover glass (Nalge Nunc International), which had been previously coated with poly-D-lysine (0.1 mg/ml) and laminin (2–5 ug/ml). The chambered cover glass was then incubated at 37°C at 5% CO2 for 15 min before 500 μl of Neurobasal media containing 2% B27, 50 ng/ml NGF, 1 μm 5-fluro-2′-deoxyuridine (Sigma), and 1μm uridine (Sigma) was added to each well.
Vincristine (0.4 μM) was added to 14 days in vitro (DIV) cultures to induce axon degeneration.
7
Isolation and Culture of Rat DRG Neurons
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According to the method described in a previous study,
21 (link) rats (2 weeks old) were used to separate dissociated DRG neurons, which were then plated in a media contained Neurobasal media plus B27 supplement, 0.5 mM L‐glutamic (Sigma‐Aldrich), penicillin (100 U/mL), and streptomycin (100 mg/mL). 3 days after plating, when more than 95% of the cells in the cultures were neurons, they were used for studies.
Acute dissociation of DRG neurons was performed as described in a previous report.
20 (link) After neurons were isolated from the L4 and L5 DRGs of adult rats and were digested, the dissociated cells were used for patch‐clamp recording within 3 to 8 h.
21 (link) rats (2 weeks old) were used to separate dissociated DRG neurons, which were then plated in a media contained Neurobasal media plus B27 supplement, 0.5 mM L‐glutamic (Sigma‐Aldrich), penicillin (100 U/mL), and streptomycin (100 mg/mL). 3 days after plating, when more than 95% of the cells in the cultures were neurons, they were used for studies.
Acute dissociation of DRG neurons was performed as described in a previous report.
20 (link) After neurons were isolated from the L4 and L5 DRGs of adult rats and were digested, the dissociated cells were used for patch‐clamp recording within 3 to 8 h.
8
Quantifying Dendritic Complexity in Hippocampal Neurons
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Live imaging and quantification of LTP were performed as described previously (Araki et al., 2015) .
Hippocampal neurons from embryonic day 18 (E18) rats were seeded on 25-mm poly-L-lysine-coated coverslips. The cells were plated in Neurobasal media (Gibco) containing 50U/ml penicillin, 50mg/ml streptomycin and 2 mM GlutaMax supplemented with 2% B27 (Gibco) and 5% horse serum (Hyclone).
At DIV 6, cells were thereafter maintained in glia-conditioned NM1 (Neurobasal media with 2mM
GlutaMax, 1% FBS, 2% B27, 1 x FDU (5mM Uridine (SIGMA F0503), 5 mM 5-Fluro-2'-deoxyUridine To obtain Sholl profiles of dendritic arbors (Sholl, 1953) , images of entire dendritic arbors of hippocampal neurons expressing DsRed were acquired and processed using Image J (Fiji) software. Scholl analysis consisted of drawing concentric rings with radii of 10, 20, 30, 40, 50, 100, and 150 μm from the center of the cell body and counting the number of dendritic intersections across each concentric circle. If a branch point fell on a line, it was counted as two crossings (Nakayama et al., 2000) .
Hippocampal neurons from embryonic day 18 (E18) rats were seeded on 25-mm poly-L-lysine-coated coverslips. The cells were plated in Neurobasal media (Gibco) containing 50U/ml penicillin, 50mg/ml streptomycin and 2 mM GlutaMax supplemented with 2% B27 (Gibco) and 5% horse serum (Hyclone).
At DIV 6, cells were thereafter maintained in glia-conditioned NM1 (Neurobasal media with 2mM
GlutaMax, 1% FBS, 2% B27, 1 x FDU (5mM Uridine (SIGMA F0503), 5 mM 5-Fluro-2'-deoxyUridine To obtain Sholl profiles of dendritic arbors (Sholl, 1953) , images of entire dendritic arbors of hippocampal neurons expressing DsRed were acquired and processed using Image J (Fiji) software. Scholl analysis consisted of drawing concentric rings with radii of 10, 20, 30, 40, 50, 100, and 150 μm from the center of the cell body and counting the number of dendritic intersections across each concentric circle. If a branch point fell on a line, it was counted as two crossings (Nakayama et al., 2000) .
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