Hibernate a media

Manufactured by Thermo Fisher Scientific

Sourced in Sweden

Hibernate A media is a culture medium designed for the preservation of cells. It maintains cell viability and minimizes metabolic activity during storage at refrigerated temperatures.

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Lab products found in correlation

5 fluoro 2 deoxyuridine, by Merck Group (1 mentions) B27 supplement, by Thermo Fisher Scientific (1 mentions) Glutamax, by Thermo Fisher Scientific (1 mentions) Poly d lysine coated coverslips, by Corning (1 mentions) Dnase, by Merck Group (1 mentions) T75 flask, by Corning (1 mentions) 6 well plate, by Greiner (1 mentions) Trypsin, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Hibernate A (26 citations) Hibernate media (7 citations)

5 protocols using hibernate a media

Isolation and Culture of Mouse Hippocampal Neurons

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Primary neurons were isolated from the hippocampi of both male and female C57Bl6/N mouse pups at post‐natal day 0 or 1. For the isolation and culture of hippocampal neurons, we followed the protocol of Beaudoin et al (2012 (link)) with slight modifications. Briefly, hippocampi were extracted into Hibernate A media (Gibco) and incubated at 37°C with papain solution for 20 min. Papain solution was removed, and trypsin inhibitor was added for 5 min. Hippocampi were then washed 3 times in pre‐warmed plating media (Neurobasal A, B27 supplement, GlutaMAX, Horse serum, 1 M HEPES pH 7.5) before being triturated 8–10 times. The suspension was strained, and 800,000 cells were seeded onto 6‐well plates coated with poly‐L‐lysine. Media was changed to neuron media (Neurobasal A, B27 supplement, GlutaMAX, Penicillin/Streptomycin) 4 h post‐seeding. Primary neurons were maintained at 37°C, 5% CO₂ or as indicated at 32°C. 5‐fluoro‐2′‐deoxyuridine (Sigma‐Aldrich) was added at a final concentration of 7.15 μg/ml to inhibit glial growth (Vossel et al, 2015 (link)). A third of the media was changed for fresh media every 4–5 days. Experimental procedures lasting 24–48 h were started at day 19–20 D.I.V. to finish at 21 D.I.V.

Preußner M., Smith H.L., Hughes D., Zhang M., Emmerichs A., Scalzitti S., Peretti D., Swinden D., Neumann A., Haltenhof T., Mallucci G.R, & Heyd F. (2023). ASO targeting RBM3 temperature‐controlled poison exon splicing prevents neurodegeneration in vivo. EMBO Molecular Medicine, 15(5), e17157.

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Dissociation of Torpid Squirrel Neurons

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Primary neurons were dissected from dorsal root ganglia of active and torpid squirrels (1 year old). Active animals were euthanized via CO₂ inhalation followed by decapitation. Dorsal root ganglia were dissected into ice-cold HBSS. Tissue was then briefly treated with collagenase P (1mg/mL in HBSS, 15 min, 37 C) followed by 0.25% trypsin (10 min, 37 C). Finally, tissue was suspended in Hibernate-A media (with custom osmolarity at 280 mOsm) supplemented with B-27 supplement and glutaMAX (GIBCO) according to the manufacturer’s recommendation (adjusted to pH 8.0 with NaOH). Hibernate-A media is designed to have a stable pH at ambient levels of CO₂. Tissue was mechanically dissociated using a plastic tipped pipette before being plated on poly-D-lysine coated coverslips (Corning) at room temperature for 30 s and immediately placed at 37 C in 0.3% CO₂, or at 10 C in 0.3% CO₂ for cold incubation. Torpid animals were euthanized by decapitation. Torpid dorsal root ganglia were dissociated identically to active tissues, but were subsequently incubated at 10 C in 0.3% CO₂. Small diameter neurons (< 30 mm) were selected for recording after becoming adherent (2hrs), for up to 36 hr after plating.

Hoffstaetter L.J., Mastrotto M., Merriman D.K., Dib-Hajj S.D., Waxman S.G., Bagriantsev S.N, & Gracheva E.O. (2018). Somatosensory Neurons Enter a State of Altered Excitability during Hibernation. Current biology : CB, 28(18), 2998-3004.e3.

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Isolation of Primary Astrocytes from Mice

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Primary astrocytes were isolated from the cortices of both male and female P1 C57BL6/N pups. Briefly, cortical tissue was extracted into Hibernate A media (GIBCO); meninges were removed and tissue was minced using a scalpel blade. The minced tissue was then incubated at 37°C for 5 minutes in fresh Hibernate A media supplemented with 1 mg/mL DNase (Sigma) and 0.25% Trypsin (GIBCO). The suspension was triturated then returned to 37°C for 5 minutes. Trituration was repeated and the suspension was incubated for a further 5 minutes. 10 mL of glial media (MEM, 10% horse serum, 0.6% D-glucose and 1% Penicillin/Streptomycin) was added and trituration was performed. The suspension was strained then centrifuged at 200 x g for 5 minutes. The cell pellet was re-suspended in glial media and seeded into T75 flasks (Corning). Cultures were maintained at 37°C, 5% CO₂ in glial media containing 1% horse serum. Upon reaching confluency, astrocytes were seeded into 6 well plates (Greiner) at a density of 100,000 cells per well.

Smith H.L., Freeman O.J., Butcher A.J., Holmqvist S., Humoud I., Schätzl T., Hughes D.T., Verity N.C., Swinden D.P., Hayes J., de Weerd L., Rowitch D.H., Franklin R.J, & Mallucci G.R. (2020). Astrocyte Unfolded Protein Response Induces a Specific Reactivity State that Causes Non-Cell-Autonomous Neuronal Degeneration. Neuron, 105(5), 855-866.e5.

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Isolation of Primary Hippocampal Neurons

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Primary neurons were isolated from the hippocampi of both male and female P1 C57BL6/N pups. Briefly, hippocampi were extracted into Hibernate A media (GIBCO) and incubated at 37°C with papain solution for 20 minutes. Papain solution was removed and trypsin inhibitor was added for 5 minutes. Hippocampi were then washed 3 times in pre-warmed plating media (Neurobasal A, B27 supplement, Glutamax, Horse serum, 1M HEPES pH 7.5) before being triturated 8-10 times. The suspension was strained and 300,000 cells were seeded onto glass coverslips coated with poly-L-lysine. Media was changed to neuron media (Neurobasal A, B27 supplement, Glutamax, Penicillin/Streptomycin) 4 hours post seeding. Primary neurons were maintained at 37°C, 5% CO₂. A third of the media was changed for fresh media every 3-4 days.

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Preparation of Rat Brain Slices

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Wistar rats 7–10 days old (P7–10) were used for this study. The animals were housed under a 12 h light/dark cycle with food and water ad libitum. All experiments were performed in accordance with the guidelines of the local ethical committee for animal research at the University of Gothenburg and were conducted in accordance with European Union directives on animal rights.
Rats were anesthetized with isofluorane and immediately decapitated. The brain hemispheres were isolated and transferred to ice cold solution (4°C) containing (in mM): 219 Glycerol, 2.5 KCl, 1.2 NaH₂PO₄, 1.2 CaCl₂, 7 MgCl₂, 25 NaHCO₃, and 11 Glucose. The solution was bubbled with carbogen gas (95% O₂ and 5% CO₂) in order to maintain the pH (7.4). Sagittal slices (300 μm) were cut using a microtome (Microtome HM 650 V, Thermo Fisher Scientific, Loughborough, UK). Slices were maintained in a Hibernate-A media (A12475-01, Gibco, Life Technologies, Sweden) at 4°C until transferred to the membrane. Slices were transferred to a membrane with hCSF and Nb-A-based culture media respectively. Slices were incubated under sterile conditions at 37°C with 5% CO₂ and 90% humidity. Culture medium was changed at regular time intervals of 2–3 days up to 9 div.

Perez-Alcazar M., Culley G., Lyckenvik T., Mobarrez K., Bjorefeldt A., Wasling P., Seth H., Asztely F., Harrer A., Iglseder B., Aigner L., Hanse E, & Illes S. (2016). Human Cerebrospinal Fluid Promotes Neuronal Viability and Activity of Hippocampal Neuronal Circuits In Vitro. Frontiers in Cellular Neuroscience, 10, 54.

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