We conducted single-cell sequencing experiments on SD rats subjected to hSYN-GFP AAV2/9 and AAV2/9-hEF1a-GFP virus sciatic nerve injection in vivo and in vitro. The rats were euthanized by cervical dislocation, and the sciatic nerve was immediately immersed in ice-cold Dulbecco’s Phosphate-Buffered Saline (DPBS, Corning). The dissected sciatic nerve was cut from the distal end of the DRG to the end of the sciatic nerve (leaving out the DRG) and we carefully peeled away the epineural sheath in cold PBS. Collected sciatic nerves were cut into pieces under a fluorescence microscope. The GFP+ piece was collected and incubated in Hibernate A (BrainBits) containing papain (100 U; Sigma) at 37°C for 2 h with intermittent flicking. After removing enzymes, the collected pieces were trypsinized for 20 min at 37°C. The tissue was triturated into an individual cell suspension using a 1 ml pipette. We removed the trypsase and cellular debris with three rounds of mild centrifugation at 1000 × g and a Hibernate A minus Ca2+ and Mg2+ wash (BrainBits). The individual cell suspension was plated into a glass-bottom plate and collected using glass pipettes under a fluorescence microscope. The glass tip was broken off and left in each PCR tube containing lysis buffer (Vazyme Biotech) with water (2.4 μl), RNase-free DNase (0.2 μl), and murine origin RNase inhibitor (0.25 μl).
Hibernate a
Manufactured by Transnetyx
Sourced in United States
Hibernate A is a laboratory equipment that provides temperature-controlled storage and incubation for biological samples. It maintains a stable and regulated environment for the preservation and study of specimens.
Automatically generated - may contain errors
Lab products found in correlation
Papain, by Merck Group (5 mentions)
Neurobasal a, by Thermo Fisher Scientific (3 mentions)
Lysis buffer, by Vazyme (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco s phosphate buffered saline dpbs, by Corning (1 mentions)
Dulbecco s phosphate buffer saline dpbs, by Corning (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Corning (1 mentions)
Picm0rg50, by Merck Group (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Glutammax, by Thermo Fisher Scientific (1 mentions)
Specific pathogen free eggs, by Charles River Laboratories (1 mentions)
Collagenase type 1, by Merck Group (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Pbs solution, by Merck Group (1 mentions)
Igf 1, by R&D Systems (1 mentions)
Alexa 488, by Merck Group (1 mentions)
Phalloidin tritc, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Laminin, by Merck Group (1 mentions)
Poly l lysin, by Merck Group (1 mentions)
22 protocols using hibernate a
1
Single-cell Sequencing of Sciatic Nerve
2
Isolation of GFP-labeled Neurons from Rat Sciatic Nerve
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Sprague-Dawley rats with hSYN or CMV-promoted GFP AAV2/9 injection in the sciatic nerve were used for electrophysiology experiments. Rats were euthanized by cervical dislocation, the sciatic nerves were immediately immersed into ice cold Dulbecco’s Phosphate-Buffer Saline (DPBS, Corning). The dissected sciatic nerve was cut from the DRG to the end of the sciatic nerve containing the trifurcation among tibial sural and peroneal branches. And then we carefully peeled away the epineural sheath in cold PBS. Collected sciatic nerves were cut into pieces under a fluorescent microscope. The GFP-positive piece was picked up and incubated in Hibernate A (BrainBits) containing papain (100 U; Sigma) at 37°C for 2 h with intermittent flicking. After the removal of enzymes, the collected pieces were trypsinized for 20 min at 37°C. The tissue was triturated into individual cell suspensions by a 1 ml pipette. Enzymes and cellular debris were removed with multiple rounds (three times) of mild centrifugation at 1000 g and washing with Hibernate A minus Ca2+ and Mg2+ (BrainBits). The individual cell suspension was plated onto a glass bottom plate.
3
Single Cell Sequencing of Sciatic Nerve
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We conducted single cell sequencing experiments on SD rats subjected to hSYN-GFP AAV2/9 and AAV2/9-hEF1a-GFP virus sciatic nerve injection in vivo and in vitro. The rats were euthanized by cervical dislocation, and the sciatic nerve was immediately immersed in ice-cold Dulbecco's Phosphate-Buffered Saline (DPBS, Corning). The dissected sciatic nerve was cut from the distal end of the DRG to the end of the sciatic nerve (leaving out the DRG) and we carefully peeled away the epineural sheath in cold PBS. Collected sciatic nerves were cut into pieces under a fluorescence microscope. The GFP + piece was collected and incubated in Hibernate A (BrainBits) containing papain (100 U; Sigma) at 37°C for 2 h with intermittent flicking. After removing enzymes, the collected pieces were trypsinized for 20 min at 37°C. The tissue was triturated into an individual cell suspension using a 1 ml pipette. We removed the trypsase and cellular debris with three rounds of mild centrifugation at 1000 ×g and a Hibernate A minus Ca 2+ and Mg 2+ wash (BrainBits). The individual cell suspension was plated into a glass-bottom plate and collected using glass pipettes under a fluorescence microscope. The glass tip was broken off and left in each PCR tube containing lysis buffer (Vazyme Biotech) with water (2.4 μl), RNase-free DNase (0.2 μl) and murine origin RNase inhibitor (0.25 μl).
4
Organotypic Hippocampal Slice Culture Protocol
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OHSC was performed based on the method described by Stoppini et al. [14 (link)] with slight modifications [13 (link),15 (link),16 (link)]. Following decapitation of mice, hippocampi were rapidly dissected, refined and then placed into a chilled dissection medium (DM) composed of hibernate A (BrainBits, Springfield, IL, USA), 2% B27 supplement, 2 mM L-glutamine by GlutamMax and antibiotic-antimycotics (all from Invitrogen, Carlsbad, CA, USA). Isolated hippocampi were sliced coronally at 300 μm thickness using a manual tissue slice chopper (#390610, Vibratome company, Saint Louis, MO, USA). The slices were gently separated from each other in fresh chilled DM and transferred onto membrane inserts (PICM0RG50; Millipore, Billerica, MA, USA) in 6-well plates containing growth medium. 4–6 slices were placed in one insert and incubated in a humidified 5% CO2 atmosphere at 37°C. The medium was entirely changed at day 1 and changed by half 3 times a week thereafter for 3–4 weeks.
To confirm effectiveness of SF medium, slices were cultured in two types of medium, which is serum-containing (SC) and SF (Table 1 ). SC medium consisted of Neurobasal A with 20% horse serum, 2 mM L-glutamine, and antibiotics-antimycotics. SF medium contained Neurobasal A with 2% B27, 2 mM L-glutamine, and antibiotic-antimycotics. After 4 days in vitro (DIV), antibiotic-antimycotics was removed from both media.
To confirm effectiveness of SF medium, slices were cultured in two types of medium, which is serum-containing (SC) and SF (
5
Marmoset Eye Embryo Collection
6
Culturing Mature Mouse Hippocampal Neurons
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Adult mouse hippocampal neurons were obtained as originally described, with modifications28 (link),29 (link). Briefly, C57Bl mice of 5–7 weeks old were killed by cervical dislocation and decapitated according to IACUC guidelines. Hippocampi were dissected out into ice-cold Ca2+-free medium Hibernate A (BrainBits, Springfield, IL), and minced into small pieces. Flushing a few times through a fire-polished Pasteur pipette further dispersed the tissue. The supernatant containing the dissociated hippocampal neurons was centrifuged at 200 g for 1 min. The cell pellet was resuspended in NbActive4 medium (BrainBitz, Springfield, IL) and seeded onto poly-L-lysine-coated glass coverslips. Hippocampal cells were incubated at 37 °C, in a wet incubator gassed with 5% CO2, 20% O2. Hippocampal neurons were kept in culture for up to two weeks with NbActive 4 medium changes every five days
7
Dissociation and Culture of DRG Neurons
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DRG neurons were dissociated and cultured as described previously25 (link), 26 (link) with modifications. Briefly, the L4-L6 DRGs were removed from the adult SD rats, and transferred to Ca2+/Mg2+-free Hibernate A (BrainBits, Springfield, IL), where the axon roots and dural tissue were manually removed. The DRGs were then transferred to 0.1% collagenase type I (Sigma, St Louis, MO). Following 1.5 h incubation at 37 °C, the DRGs were dissociated in 0.25% trypsin (Gibco) for an additional 15 min at 37 °C, and mechanically triturated through a pipette into the single cell suspension. To remove SCs, a partial purification step was performed by centrifugation at 900 rpm for 5 min on 15% BSA in PBS solution (Sigma). The obtained DRG neurons were cultured on the coated plates in Neurobasal-A and B-27 minus insulin (Gibco) supplemented with penicillin–streptomycin (both 50 U/ml, Gibco). The modulatory effects of IGF-1 on neurite outgrowth were treated with IGF-1 (25 ng/ml; R&D Systems) for 48 h. For the pre-lesion injury assay, we transected the sciatic nerve, waited 4 days, and then cultured adult DRG neurons for 72 h to evaluate their regeneration capacity.
8
Retinal Explant Visualization in PTEN Mutant Mice
9
Isolation and Culture of Dopaminergic Neurons
10
Cell Culture and Imaging Techniques
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COS-7, U2-OS, and HeLa cells (Cell Culture Facility, UC-Berkeley) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco 31053-028) supplemented with 10% fetal bovine serum (Corning), 1× GlutaMAX Supplement, and 1× non-essential amino acids, at 37 °C and 5% CO2. For live-cell experiments, cells were cultured in Lab-Tek 8-well chambered coverglass (ThermoFisher), and transiently transfected with the above plasmids, either alone or in combination. Transfection was performed using Lipofectamine 3000 (ThermoFisher) or the Neon Transfection System (ThermoFisher), following the manufacturers’ instructions. For live-cell staining of the plasma membrane, lipid droplet, and DNA, wheat germ agglutinin (WGA) CF532 (300–500×, 29064, Biotium), LipidSpot488 (300–1000×, 70065, Biotium), and SYBR Green (15,000–100,000×, S7536, ThermoFisher) were added to the medium for 30 min at 37 °C and washed three times with DMEM before imaging. Imaging buffer was the regular culture medium with the addition of 25 mM HEPES at pH 7.4 (15630106, Gibco) or a commercial buffer based on MOPS (Hibernate A, BrainBits), with similar results observed.
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