Nov 29, 2024
Mouse primary cortical and hippocampal neuron preparation
- 1California Institute of Technology
Protocol Citation: Gerard Michael Coughlin 2024. Mouse primary cortical and hippocampal neuron preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv52925v1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 28, 2024
Last Modified: November 29, 2024
Protocol Integer ID: 113105
Keywords: Mouse primary neurons, Primary culture, Cortical neurons
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020495
Abstract
This protocol describes preparation of cortical and hippocampal neurons from E15-E17 mouse embryos. These neurons may be maintained for at least 2 weeks. This in vitro model may be useful for studying neuronal physiology, viral transduction, or tool development.
*IMPORTANT*
This protocol is heavily adapted from https://app.jove.com/v/54981/reliable-identification-living-dopaminergic-neurons-midbrain-cultures. Review and watch the referenced protocol for greater insight into the procedures described.
Materials
Reagents
HBSS, no calcium, no magnesium, no phenol redThermo Fisher ScientificCatalog #14175095
Papain from papaya latexMerck MilliporeSigma (Sigma-Aldrich)Catalog #P3125-250MG
HyClone donor equine serum, U.S. originCytivaCatalog #SH30074.03
Bovine Serum Albumin (BSA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A7030-100G
DPBS with no calcium and magnesiumThermo Fisher ScientificCatalog #14190-144
Laminin Mouse Protein, NaturalThermo FisherCatalog #23017015
Poly-L-Ornithine Merck MilliporeSigma (Sigma-Aldrich)Catalog #P4957
POLY-D-LYSINE HYDROBROMIDE MOL WT 70000 - 5MGMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6407-5MG
BrainPhys™ Neuronal MediumSTEMCELL Technologies Inc.Catalog #05790
NeuroCult™ Neuronal Plating MediumSTEMCELL Technologies Inc.Catalog #05713
NeuroCult™ SM1 Neuronal Supplement 10 mL
STEMCELL Technologies Inc.Catalog #5711
UltraPure™ DNase/RNase-Free Distilled WaterThermo Fisher ScientificCatalog #10977023
Glutamax (100x)Gibco - Thermo FischerCatalog #35050-061
L-Glutamic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #G1251-100G
Consumables
0.2 µm sterile syringe filters (e.g. Millex-GP Syringe Filter Unit, 0.22 µmMerck Millipore (EMD Millipore)Catalog #SLGP033RS )
Filtered pipette tips, including P1000 tips
Sterile 1.5 mL, 15 mL, and 50 mL tubes
Glass coverslips, if desired (e.g. High performance coverslip 12mm dia 1.5h treated sterile super resolutionNeuvitro CorporationCatalog #GG-12-1.5h-pre )
Glass bottomed dishes, if desired (e.g. µ-Plate 24 Well Glass BottomIbidiCatalog #82427 )
Sterile, non-TC treated 24-well plates
Sterile, 10 cm petri dishes for dissection
Sterile bottle or tube filters (e.g. Steriflip-GP Sterile Centrifuge Tube Top Filter UnitMerck Millipore (EMD Millipore)Catalog #SCGP00525
)
Tools
4 pairs of fine-nosed forceps
2 pairs of dissecting scissors
No. 10 scalpel blade
Equipment
2 Chilled blocks or 2 ice buckets, with ice
Certified biosafety cabinet
Laminar flow hood
Bead sterilizer
Stereomicroscope and light source
Carbon dioxide euthanasia station
Reagent preparation
Reagent preparation
General note on reagent preparation:
Note
Prepare all reagents with sterile technique, and if possible, in a BSC.
Prepare poly-D-lysine solution 0.1 mg/mL , by adding 50 mL of UltraPure water to 5 mg bottle of poly-D-lysine. Invert to mix and allow dissolution by leaving it at 4 °C Overnight .
Solution can be kept at 4 °C for at least a month.
Prepare laminin working solution 0.02 mg/mL , by diluting laminin in appropriate volume of UltraPure water. Prepare enough for 200 µL per coverslip or well.
Prepare immediately before use.
Note
Thaw laminin slowly at 4 °C to prevent gelation. Upon receiving, we thaw then aliquot laminin to appropriate volumes, and store at -20 °C .
Prepare papain solution. Dilute papain to 15 units/mL in 1x HBSS. Pipette up and down 5 times to mix thoroughly.
Prepare on the day of dissection. For 4 cortical hemispheres (2 embryos), you will need 1 mL of papain solution.
Prepare stop solution. Dilute 1 mL donor equine serum to 10% in 9 mL 1x HBSS. Sterile filter with a 0.2 µm syringe filter tip. Store at 4 °C .
Prepare on the day of dissection.
Prepare 4% Bovine Serum Albumin (BSA) solution. Weigh out 200 mg of BSA and add 1x DPBS to a total final volume of 5 mL . Filter sterilize with 0.2 µm syringe filter tip. Store at 4 °C.
Prepare on the day of dissection.
Prepare complete maintenance media. Combine BrainPhys media and SM1 supplement. For 50 mL of complete media, combine 49 mL of BrainPhys media with 1 mL of SM1 supplement. Sterile filter.
Complete maintenance media can be stored in 4 °C for up to 1 month.
Prepare L-glutamic acid stock solution. Dissolve L-glutamic in UltraPure water to a concentration of 2 mg/mL . Sterile filter, aliquot to appropriate volumes and store at -20 °C .
Prepare plating media. Combine NeuroCult plating media, SM1 supplement, Glutamax, and L-glutamic acid. For 10 mL of plating media, combine 9.8 mL of plating media, 200 µL of SM1 supplement, 25 µL of Glutamax, and 18.5 µL of L-glutamic acid. Sterile filter and store at 4 °C .
Prepare up to a few days before prep.
Coating coverslips or plate
Coating coverslips or plate
3m
3m
General note on coating:
Note
We found that coating glass wells or coverslips with poly-D-lysine, poly-L-ornithine and laminin yielded consistently healthy primary cultures. Other coating formulations may work better in your hands.
Prepare coating and wash steps in a BSC. Begin coating 3 days before planned dissection.
(If using coverslips) In a sterile manner, transfer 1 coverslip to each well of a non-TC-treated 24 well plate.
Coat coverslips or well bottom with poly-D-lysine. Pipette 200 µL of poly-D-lysine solution (0.1 mg/mL ). Incubate at 37 °C Overnight in a tissue culture incubator.
Aspirate poly-D-lysine solution. Perform 6 washes with UltraPure water, for 00:03:00 per wash.
Pipette 200 µL of poly-L-ornithine onto coverslip or well bottom. Incubate at 37 °C Overnight in a tissue culture incubator.
Aspirate poly-L-ornithine solution. Perform 3 washes with UltraPure water, for 00:03:00 per wash.
Pipette 200 µL of laminin onto coverslip or well bottom. Incubate at 37 °C Overnight in a tissue culture incubator.
Aspirate laminin solution. Perform 3 washes with DPBS, for 00:03:00 per wash.
Pipette 300 µL of complete plating medium onto coverslip or well bottom. Incubate at 37 °C in a tissue culture incubator until use.
3m
Prepare solutions and spaces
Prepare solutions and spaces
Prepare ice-cold 1x HBSS for dissection and papain solution. For 4 cortical hemispheres isolated from 2 embryos, you will need 20 mL of ice-cold 1x HBSS and 1 mL of papain solution. Keep both on ice until use.
Pre-warm plating media by transferring into a tissue culture incubator.
Note
Pre-warming media in an incubator allows the media to become appropriately buffered. Ensure that you thoroughly disinfect the media tube before placing into incubator.
Sterilize tools using bead sterilizer and disinfect the laminar flow hood.
Prepare 2 cooling blocks: one for holding the embryos still in utero and one for dissection of the cortex and further sectioning.
Prepare 4 petri dishes: one for embryos in uterus, one for dissecting tissue, one for the cortex and hippocampus post-dissection, and one for resting the sterile tools in.
Collect scalpel blade, P1000 pipette and sterile scissors to make wide-bore pipette tips.
Tissue collection
Tissue collection
Euthanize a timed pregnant mouse on E15-E17 using CO2.
Sterilize the abdomen of euthanized mouse with 70% ethanol. Using surgical scissors, open the abdominal cavity and fold the abdominal wall over with forceps to expose the abdominal cavity.
Remove the uterus by cutting both ends of the uterine horn. Place the tissue into a Petri dish on a chiller. Transfer chiller and Petri dish to sterile laminar flow hood.
With forceps in each hand, open the embryo sac and remove the embryo. Decapitate the embryo using the scalpel blade. Position the head dorsal side up.
Using forceps in one hand, firmly grasp the snout to stabilize the tissue.
Using the forceps held in the other hand, gently remove the skin. Then using forceps, peel the skull off caudally along midline. Ensure that skull is adequately removed to allow for removal of the brain.
Once the brain is exposed, remove the brain from the skull.
Position forceps such that one tip is between the cortex and mesencephalon and the other is over the cerebellum. Gently grasp and lift out the entire brain, then place it in a Petri dish with ice-cold 1x HBSS, under a dissecting stereomicroscope and with adequate lighting.
Note
You may also remove the brain by gently overturning the skull and brain above the petri dish with ice-cold 1x HBSS, and teasing the brain out with forceps.
Repeat the procedure starting with step 21 for the remaining embryos.
Once all brains are collected, proceed with microdissection.
Under the dissecting microscope, orient the brain so that the dorsal side is accessible. Slip one set of forceps under either edge of the cortex, pinch and flip it away from the rest of the brain, leaving the hippocampus intact. Remove meninges and vasculature by gently grasping the meninges and pulling upward away from the brain. Pinch off olfactory bulb with forceps. Transfer the cortex with hippocampus to a clean petri dish with ice-cold 1x HBSS.
Repeat microdissection for remaining brains.
After all the brains have been microdissected, use forceps and a No. 10 scalpel to section each cortex into 8 pieces of approximately equal size.
Cell dissociation
Cell dissociation
Sterilely cut the tip off of a P1000 pipette tip, and use the tip to transfer the microdissected cortices and hippocampi to a 15 mL conical tube. Once the tissue has settled at the bottom, remove excess HBSS.
Transfer tube to sterile BSC.
Add 1 mL of papain solution to the tissue, and transfer to a 37 °C waterbath. Incubate for 00:15:00 , gently mixing the suspension by tapping halfway through.
Sterilely cut the tip off of a P1000 pipette tip.
Once the papain incubation is finished, use the wide bore tip to transfer the tissue from the papain solution into a new 15 mL conical tube with 1 mL of ice-cold stop solution.
Once the tissue has settled at the bottom, remove excess stop solution, then add 1 mL of fresh ice-cold stop solution. Repeat this rinse step 1 more time.
Create a single cell suspension by gently titurating cells with a P1000 pipette. Pipette cell suspension up and down 8 times.
Note
Avoid over tituration, as this may lead to poor cell viability.
Using P1000 pipette, transfer 800 µL of cell suspension to a new 15 mL conical tube. Avoid collecting any undissociated tissue segments. Underlay 200 µL of 4% BSA solution by slowly pipetting BSA into the bottom of the tube. Slowly and carefully withdraw the pipette tip to avoid disrupting the cell suspension.
Centrifuge the suspension at 280 x g, 18°C, 00:06:00 , then aspirate the supernatant with a P1000. Resuspend the cells in 1 mL of plating medium.
Count cells using a hemocytometer and further dilute to desired concentration with plating medium.
Note
We prefer to resuspend to the final concentration that will be in the well. For example, if you want to plate 60,000 cells in 300 µL , resuspend cells to a concentration of 200 cells/µL .
Plate cells. Aspirate plating media from wells/coverslips, and add appropriate volume of diluted cells.
Maintain the culture. We leave cells in plating medium for 5 days, then perform a half media change with complete maintenance media (alternatively, add equal volume of complete maintenance media). After this point, do a half media change every 3-4 days.
Protocol references
Henley, B. M., Cohen, B. N., Kim, C. H., Gold, H. D., Srinivasan, R., McKinney, S., Deshpande, P., Lester, H. A. Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression. J. Vis. Exp. (120), e54981, doi:10.3791/54981 (2017).