Hibernate media

Manufactured by Thermo Fisher Scientific

Sourced in United States

Hibernate media is a specialized cell culture medium designed to maintain cells in a dormant state during storage or transportation. It provides the necessary nutrients and conditions to keep cells viable while minimizing metabolic activity and growth.

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Hibernate-E media (6 citations) Hibernate A media (5 citations)

7 protocols using hibernate media

Embryonic Nasal Tissue Cryosectioning and RNA Extraction

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Ethical approval for the use of postmortem human foetal nasal tissue was given by the National Research Ethics Service Committee East of England - Cambridge Central, UK. The tissue was collected at Addenbrooke’s Hospital (Cambridge, UK) from patients who had requested pregnancy terminations, and dissected at the John van Geest Centre for Brain Repair, University of Cambridge, Cambridge, UK. Tissue comprising the cartilage of the nasal septum, the medial zygomatic region, the maxillae of the developing upper jaw, and the base of the orbital cavities was dissected in HIBERNATE media (Gibco) from the facial region of 7-8-week embryos.
For cryosectioning, the tissue was immersion-fixed overnight in 4% paraformaldehyde in PBS at 4°C, then cryoprotected in 30% diethylpyrocarbonate-treated sucrose, embedded in O.C.T. compound (Tissue Tek), flash frozen in isopentane on dry ice and stored at -80°C. 10 μm sections were taken on a rotary cryostat (Bright Instrument Company).
For cDNA synthesis, the tissue was transferred to the lysis buffer from the Arcturus PicoPure RNA extraction kit (ThermoFisher Scientific), and then to Trizol (Invitrogen) for homogenisation and total RNA extraction according to the manufacturer's instructions. Single-strand cDNA was generated using Invitrogen’s Superscript III First-Strand Synthesis System kit.

Miller S.R., Perera S.N., Benito C., Stott S.R, & Baker C.V. (2016). Evidence for a Notch1‐mediated transition during olfactory ensheathing cell development. Journal of Anatomy, 229(3), 369-383.

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Isolation of Human Fetal Nasal Tissue

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Ethical approval for the use of post mortem human foetal nasal tissue was given by the National Research Ethics Service Committee East of England – Cambridge Central, UK. The tissue was collected at Addenbrooke's Hospital (Cambridge, UK) from patients who had requested pregnancy terminations, and dissected at the John van Geest Centre for Brain Repair, University of Cambridge, Cambridge, UK. Tissue comprising the cartilage of the nasal septum, the medial zygomatic region, the maxillae of the developing upper jaw and the base of the orbital cavities was dissected in HIBERNATE media (Gibco) from the facial region of 7–8‐week embryos.
For cryosectioning, the tissue was immersion‐fixed overnight in 4% paraformaldehyde in PBS at 4 °C, then cryoprotected in 30% diethylpyrocarbonate‐treated sucrose, embedded in O.C.T. compound (Tissue Tek), flash frozen in isopentane on dry ice and stored at −80 °C. Ten‐micrometre sections were taken on a rotary cryostat (Bright Instrument Company).
For cDNA synthesis, the tissue was transferred to the lysis buffer from the Arcturus PicoPure RNA extraction kit (ThermoFisher Scientific), and then to Trizol (Invitrogen) for homogenisation and total RNA extraction according to the manufacturer's instructions. Single‐strand cDNA was generated using Invitrogen's Superscript III First‐Strand Synthesis System kit.

Miller S.R., Perera S.N., Benito C., Stott S.R, & Baker C.V. (2016). Evidence for a Notch1‐mediated transition during olfactory ensheathing cell development. Journal of Anatomy, 229(3), 369-383.

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Primary Cortical Neuron Culture Protocol

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Primary dissociated cortical cultures were prepared as described previously (Atkinson et al., 2015) with slight modifications. Heterozygous mice were mated and resulting embryos were harvested at embryonic day (E) 15.5. Each embryo was cultured individually and liver tissue was collected for genotyping as outlined in above. Following decapitation, heads were stored in Hibernate media (Gibco) at 4°C until time of culture (10 minutes to 1 hour), with samples blinded to the researcher until the end of experimental analysis. Cortical tissue (including both cortex and hippocampus) was collected into 1ml HBSS and enzymatically dissociated in 0.0125% trypsin for 4 minutes, prior to plating. Cells were plated onto a variety of surfaces, pre-coated with 10% poly L-lysine (Sigma Aldrich). For immunofluorescence and neurite outgrowth assays, cells were plated onto 12mm coverslips at a concentration of 30,000 viable cells per coverslip. For protein harvest, whole cells were plated into 12 well trays at a concentration of 200,000 viable cells per well.

Atkinson R.A.K., Fair H.L., Wilson R., Vickers J.C, & King A.E. (2021). Effects of TDP-43 overexpression on neuron proteome and morphology in vitro. Molecular and cellular neurosciences, 114.

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Isolation and Culture of Murine Neural Progenitor Cells

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At postnatal day one of life (P1), pups (aldh5a1^+/+ and aldh5a1^−/−) were sacrificed, and the brain gently eviscerated into Hibernate media supplemented with penicillin/streptomycin and B27 medium (Thermo Fisher, Waltham, MA, USA). The cortex and hippocampal regions were dissociated with Versene (Thermo Fisher) in media (37°C). Cells were maintained in complete medium consisting of Neurobasal^® A Medium (Thermo Fisher) supplemented with B-27^® Serum-Free Supplement, GlutaMAX™ (Gibco, Thermo Fisher), antibiotic-antimycotic solution (Gibco, Thermo Fisher), FGF-basic (recombinant human (bFGF)), and EGF (recombinant human epidermal growth factor; Millipore, Darmstadt, Germany). Cells were expanded in 35-mm and 100-mm dishes coated with poly-L-ornithine (Sigma, St. Louis, MO). Confluent cells were passaged using Versene. Tail biopsies from P1 pups were obtained and genotype was determined as described (Hogema et al 2001 (link)). The neuronal status of these cells was confirmed using Nissl staining, as well as immunohistochemistry for Nestin and Sox2.

Vogel K., Ainslie G., McConnell A., Roullet J.B, & Gibson K. (2017). Toxicologic/Transport Properties of NCS-382, a γ-Hydroxybutyrate (GHB) Receptor Ligand, in Neuronal and Epithelial Cells: Therapeutic Implications for SSADH Deficiency, a GABA Metabolic Disorder. Toxicology in vitro : an international journal published in association with BIBRA, 46, 203-212.

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Human Fetal Tissue Collection Protocol

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Human fetal tissues were collected from 6-11 weeks PC legally terminated embryos at Malmö Hospital (Malmö, Sweden) and Addenbrooke's Hospital (Cambridge, UK). Ethical approval for the use of postmortem human fetal tissue was provided by the Swedish National Board of Health and Welfare under existing guidelines, including informed consent from women seeking abortions, and by the National Research Ethics Service Committee East of England – Cambridge Central (Local Research Ethics Committee, 96/085). The gestational age of each embryo was determined by crown-to-rump length (CRL) (mm) measured at either the time of dissection when the quality of the embryo allowed for this or otherwise estimated by ultrasound measurements before abortion. The external features of the embryo were also carefully monitored to confirm that the CRL correlated with the appropriate embryonic stage. Samples from the UK were shipped overnight on ice in Hibernate media (ThermoFisher Scientific) to Sweden.

Birtele M., Storm P., Sharma Y., Kajtez J., Wahlestedt J.N., Sozzi E., Nilsson F., Stott S., He X.L., Mattsson B., Ottosson D.R., Barker R.A., Fiorenzano A, & Parmar M. (2022). Single-cell transcriptional and functional analysis of dopaminergic neurons in organoid-like cultures derived from human fetal midbrain. Development (Cambridge, England), 149(23), dev200504.

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Isolation and Flow Cytometry of Retinal Myeloid Cells

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Cells were harvested and counted as described previously [15 (link)] with some modifications. Briefly, two retinas were minced in 1 ml of Hank’s Balanced Salt Solution (Thermo Fisher Scientific) containing 2 mg/ml Collagenase D and 28 U/ml DNase I (both from Sigma-Aldrich). Tissue was incubated at 37 °C for 60 min, and re-suspended in 2 ml of Hibernate media (Thermo Fisher Scientific). The suspension was filtered through a 140 μm-mesh screen (Sigma-Aldrich), washed, and centrifuged; supernatant was carefully removed. Cell pellets were fixed and permeabilized for 10 min at RT in 4% paraformaldehyde/0.1% saponin buffer (Sigma-Aldrich), then blocked for 15 min at RT in 3% BSA. Cells were incubated in Alexa Fluor 647 conjugated anti-mouse CD11b antibody (1:100, Biolegend, USA) and Alexa Fluor 488 conjugated IB4 (1:100, Thermo Fisher Scientific) overnight at 4 °C. Suspensions were finally washed and re-suspended in 300 μl PBS. Data were acquired on a BD FACScan flow cytometer (BD Biosciences), 10,000 events were collected for each sample and then analyzed using the FlowJo software (Tree Star).

Wang X., Miller E.B., Goswami M., Zhang P., Ronning K.E., Karlen S.J., Zawadzki R.J., Pugh EN J.r, & Burns M.E. (2017). Rapid monocyte infiltration following retinal detachment is dependent on non-canonical IL6 signaling through gp130. Journal of Neuroinflammation, 14, 121.

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Culturing Primary Hippocampal Neurons from Rats

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Primary cultures were prepared from P0/P1 rat pups (Sprague-Dawley) as described earlier (Goslin and Banker, 1989 (link)). The hippocampus was dissected out from the rodent brain in Hibernate Media (Thermofisher Scientific). The dissected hippocampi were trypsinized (0.25% Trypsin), washed 3×, and dissociated into single cells by syringing using needles of 2 pore sizes sequentially to remove all neurites (21 G and 25 G, BD Precision Glide Needle). The cells were plated on 18 mm diameter thickness corrected coverslips (Marienfeld Superior) coated with Poly-L-Lysine (1 mg/ml, Sigma Aldrich) after resuspending in Neurobasal media (Thermofisher Scientific) complemented with B27, Glutamate, and Normocin (Invivogen; referred to as Complete Neurobasal media) at a density of 50 cells/mm².

Jose M., Sivanand A, & Channakeshava C. (2021). Membrane Cholesterol Is a Critical Determinant for Hippocampal Neuronal Polarity. Frontiers in Molecular Neuroscience, 14, 746211.

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