Organoid tissue slices (two slices from an H9 day 53 organoid then cultured at the ALI for 22 days; and two slices from each of two H1 day 53 organoids then cultured at the ALI for 16 days) were transferred from the ALI into a conical tube (the two slices from the same organoid were pooled) containing Hibernate Medium (Thermo Fisher Scientific, A1247601) plus 1X B-27 Supplement (Thermo Fisher Scientific, 17504044). Gentle dipping helped remove any remaining agarose. The slices were then placed into a 10cm2 tissue culture dish and after washing twice with 1X dPBS (Sigma, D8537), transferred into a gentleMACS C Tube (Miltenyi, 130-093-237) containing 2 ml of Accumax (Sigma, A7089) solution. The C Tube was subsequently attached to the gentleMACS Octo Dissociator (Miltenyi) and ran with the recommended cell dissociation settings. The cell suspension was run through a 70μm strainer to remove any residual cell and debris clumps. A small volume of the strained cell suspension was removed for cell counts with the remaining being diluted 4-fold in 1X dPBS and then centrifuged at 200g for 5 minutes. The cell pellet was then resuspended in dPBS containing 0.04% BSA (Sigma, A9418) to give a final concentration of 206 cells/μl. This suspension was kept on ice for 30 minutes until being processed.
Hibernate medium
Manufactured by Thermo Fisher Scientific
Sourced in Australia
Hibernate Medium is a laboratory product designed for the cryogenic preservation of cells, tissues, and other biological samples. It provides a specialized environment to maintain the viability and functionality of these samples during long-term storage in liquid nitrogen or other cryogenic conditions.
Automatically generated - may contain errors
Lab products found in correlation
Papain, by Merck Group (3 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Merck Group (2 mentions)
Gentlemacs c tube, by Miltenyi Biotec (2 mentions)
Accumax, by Merck Group (2 mentions)
Gentlemacs octo dissociator, by Miltenyi Biotec (2 mentions)
Optiprep, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Cd11b conjugated magnetic beads, by Miltenyi Biotec (1 mentions)
Dnase, by Roche (1 mentions)
Collagenase, by Worthington (1 mentions)
Glutamine, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Neurobasal a b27, by Thermo Fisher Scientific (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Dnase, by Merck Group (1 mentions)
Brainphys, by STEMCELL (1 mentions)
6 protocols using hibernate medium
1
Dissociation of Organoid Tissue Slices
2
Dissociation of Organoid Tissue Slices
3
Cortical Neuron Isolation and Co-culture with Astrocytes
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Twenty-four hours after the mechanical trauma, cortical neurons were isolated from P5–6 mice and plated on top of the reactive astrocytes as previously reported7 (link), 8 (link), 35 (link). Cortices were dissected in Hibernate medium (Life Technologies) and incubated for 30 min at 30 °C with 2 mg/ml papain (Sigma-Aldrich, Australia). After enzymatic digestion, cells were dissociated by gentle pipetting and the cellular suspension was purified by Optiprep (Sigma-Aldrich) gradient to isolate neurons from the other cells as previously reported7 (link), 8 (link), 35 (link). Dissociated cortical neurons were plated at a density of 20,000/well on top of pre-prepared astrocyte cultures.
All pharmacological treatments were performed for 24 hrs in medium containing 0% FBS. PEG-IGF-I was diluted in PBS and used at a final concentration of 100 ng/mL.
All pharmacological treatments were performed for 24 hrs in medium containing 0% FBS. PEG-IGF-I was diluted in PBS and used at a final concentration of 100 ng/mL.
4
Isolation and Culture of Cortical Neurons from Mice
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Twenty-four hours after the mechanical trauma, cortical neurons were isolated from P5-6 mice and plated on top of the reactive astrocytes as previously reported13 (link)14 (link). Cortices were dissected in Hibernate medium (Life Technologies) and incubated for 30 min at 30 °C with 2 mg/ml papain (Sigma-Aldrich, Australia). After enzymatic digestion, cells were dissociated by gentle pipetting and the cellular suspension was purified by Optiprep (Sigma-Aldrich) gradient to isolate neurons from the other cells as previously reported13 (link)14 (link). Cortical neurons thus dissociated were plated at a density of 20,000/well on the top of pre-prepared astrocyte cultures.
5
Isolation of Post-Mortem Microglia
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Microglia were isolated from post-mortem brain tissue as described previously39 (link). After autopsy, tissue was stored in Hibernate medium (Invitrogen, Carlsbad, CA, USA) at 4 °C until further processing. Microglia isolation started as soon as possible, at the latest after 24 h. A single-cell suspension was generated by mechanical and enzymatic digestion with collagenase (3700 units/mL; Worthington, USA) and DNase (200 µg/mL; Roche, Switzerland) for frontal lobe (GFM), temporal lobe (GTS) and thalamic (THA) tissues, or 0.2% trypsin and 30 mg DNase for subventricular zone (SVZ) tissue. A Percoll (Amersham, Merck, Germany) gradient was generated to separate viable cells from myelin, cellular debris, and erythrocytes. The middle layer was collected and washed twice, followed by positive selection of myeloid cells with CD11b-conjugated magnetic beads (Miltenyi Biotec, Germany) according to the manufacturer’s protocol. MACS-isolated CD11b+ cells were fixed with fixation/stabilization buffer (SmartTube) and frozen at −80 °C until analysis by mass cytometry39 (link).
6
Murine Primary Cortical Neuron Culture
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Primary cultures from P1 rat and mouse cortex were prepared similar as previously described (Moutin et al., 2020) (link). Animals from both sexes were used. After removal of meninges, the neocortex was dissected and enzymatically digested with Papain (Sigma) in the presence of DNAse (Sigma), followed by mechanical dissociation and centrifugation through a cushion of 4% bovine serum albumin (BSA; Sigma). These steps were completed using Hibernate medium (Invitrogen). Cells were then plated onto poly-D-lysine (Sigma) coated coverslips in 24-well plates. For each coverslip, 25,000 cells were allowed to settle in a 40 µl drop for 30 min and then each well was filled with 500 µl growth medium: Neurobasal-A/B27 (1:50, Invitrogen) supplemented with GlutaMax (1:400, Invitrogen), glutamine (0.25-0.5 mM, Sigma), penicillin/streptomycin (1:100, ThermoFisher), and heat-inactivated fetal calf serum (10%, Sigma). Medium was partially exchanged on Day 3 (480 µl) and Day 7 (100 µl) with fresh maintenance medium: BrainPhys (StemCell), B27 (1:50, Invitrogen), GlutaMax (1:400, Invitrogen), and penicillin/streptomycin (1:100, ThermoFisher). Cultures were maintained for up to 3 weeks without any further medium change at 37°C and 5% CO 2 .
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