May 23, 2023
Primary neuron culture for live imaging of axonal cargoes
- C. Alexander Boecker1,
- Erika L.F. Holzbaur2,3
- 1Department of Neurology, University Medical Center Goettingen, 37077 Goettingen, Germany;
- 2Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA;
- 3Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, USA
External link: https://doi.org/10.1016/j.celrep.2023.112448
Protocol Citation: C. Alexander Boecker, Erika L.F. Holzbaur 2023. Primary neuron culture for live imaging of axonal cargoes. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgby723vpk/v1
Manuscript citation:
Dou D, Smith EM, Evans CS, Boecker CA, Holzbaur EL, Regulatory imbalance between LRRK2 kinase, PPM1H phosphatase, and ARF6 GTPase disrupts the axonal transport of autophagosomes. Cell reports 42(5). doi: 10.1016/j.celrep.2023.112448
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: October 11, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 71157
Keywords: Dissection, Mouse, Primary neuron, Live-imaging, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000350
Abstract
This protocol describes the preparation and culture of mouse primary cortical neurons for live-imaging experiments. Cortices were dissected from mouse embryos at day 15.5. Cortical neurons were isolated by digestion with 0.25% Trypsin and trituration with a serological pipette. Neurons were plated on glass-bottom imaging dishes in Attachment Media. After 5 hours in culture, Attachment Media was replaced with Maintenance Media, and AraC was added on the next day to prevent glia cell proliferation. Neurons were transfected 16-24 hours before imaging using Lipofectamine 2000.
Attachments
547-1143.pdf
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Materials
Materials:
- Dissecting microscope
Equipment
Dumont #5 - Mirror Finish Forceps
NAME
Forceps
TYPE
Durmont
BRAND
11251-23
SKU
LINK
- Micro spring scissors (Fine Science Tools)
Equipment
Countess 3 FL Automated Cell Counter
NAME
Automated Cell Counter
TYPE
Thermofisher scientific
BRAND
AMQAF2000
SKU
LINK
Equipment
35 mm Dish | No. 1.5 Coverslip | 20 mm Glass Diameter | Uncoated
NAME
Coverslip
TYPE
Mattek
BRAND
P35G-1.5-20-C
SKU
LINK
- 15 mL conical tubes
- 10 cm cell culture dish
Reagents:
- Poly-L-lysine hydrobromideSigma AldrichCatalog #p1274
- 10X HBSSThermo Fisher ScientificCatalog #14185-052
- HEPES BufferThermo Fisher ScientificCatalog #15630-080
- Trypsin (2.5%), no phenol redThermo FisherCatalog #15090046
- MEMThermo FisherCatalog #11095072
- Horse serumThermo Fisher ScientificCatalog #16050122
- 45% D-( )-GlucoseSigmaCatalog #G8769
- Sodium Pyruvate (100 mM)Thermo Fisher ScientificCatalog #11360070
- Trypan Blue Stain (0.4%) for use with the Countess™ Automated Cell CounterThermo FisherCatalog #T10282
- Neurobasal™ MediumThermo FisherCatalog #21103049
- B-27™ Supplement (50X), serum freeGibco - Thermo FisherCatalog #17504044
- GlutaMAX™ SupplementThermo FisherCatalog #35050061
- Penicillin-Streptomycin (10,000 U/mL)Thermo Fisher ScientificCatalog #15140122
- Cytosine β-D-arabinofuranoside hydrochloride (AraC)Sigma AldrichCatalog #C6645
- Lipofectamine™ 2000 Transfection ReagentThermo Fisher ScientificCatalog #11668019
- Hibernate E Low Fluorescence Imaging Medium (BrainBits, Cat# HELF)
Safety warnings
Take necessary precautions with sharp objects during dissection. Follow institutional recommendations for disposal of animal tissue and biohazardous materials.
Day before dissection
Day before dissection
Coat glass-bottom imaging dishes with PLL.
Hydrate 100 mg PLL (Sigma) in 50 mL 0.1 Molarity (M) borate buffer, 8.5 .
Store PLL stock solution (2 mg/mL ) in 1 mL aliquots at -80 °C .
On the day before neuron dissection, dilute PLL in ddH2O 1:20 to a final concentration of 100 μg/ml .
Add 1 mL PLL to each glass-bottom imaging dish (MatTek) and incubate Overnight at 37 °C .
Only coat the glass center with PLL.
Note
For easy handling, we find it helpful to place imaging dishes in 10 cm or 15 cm cell culture dishes.
Prepare HBSS, attachment media and maintenance media.
For 500 mL 1x HBSS, combine
| A | B | |
| 10 x HBSS | 50 mL | |
| 1 M HEPES | 5 mL | |
| ddH2O | up to 500 mL | |
| Filter-sterilize | ||
Store 1x HBSS at 4 °C and use within one month.
For 50 mL attachment media, combine
| A | B | |
| Heat-inactivated horse serum | 5 mL | |
| 100 mM Sodium pyruvate | 500 µL | |
| 45% Glucose | 660 µL | |
| MEM | up to 50 mL |
For 50 mL maintenance media, combine
| A | B | |
| GlutaMAX | 500 µL | |
| Penicillin/Streptomycin | 500 µL | |
| 45% Glucose | 660 µL | |
| B-27 | 1 mL | |
| Neurobasal | Up to 50 mL |
Store attachment media and maintenance media at 4 °C .
Note
Maintenance Media should be used within 7 days. Attachment media can be kept at 4 °C for 3-4 weeks.
Dissection of cortical neurons
Dissection of cortical neurons
In the morning of the day of dissection, wash PLL-coated imaging dishes twice with sterile ddH2O.
Add 2 mL attachment media per imaging dish and leave dishes at 37 °C in cell culture incubator.
Warm required amount of attachment media and 1x HBSS (4.5 mL for one dissection) in 37 °C water bath.
Aliquot maintenance media into 10 cm cell culture dish to equilibrate in 37 °C / 5% CO2 cell culture incubator.
Let 2.5% trypsin aliquots thaw at Room temperature .
Sacrifice pregnant mouse, dissect embryos, and place embryonic brains in HBSS On ice .
Using a dissecting microscope, remove meninges from brain hemispheres with fine forceps.
Isolate cortices using fine forceps and small spring scissors.
Transfer dissected cortices into a 15 mL conical tube filled with 5 mL HBSS and keep On ice until all cortices are collected.
Note
Use clean and sterile equipment for all dissection steps to prevent bacterial contamination of neuron cultures.
Note
We find that using ice-cold HBSS helps preventing the tissue from getting sticky during the dissection. If HBSS gets too warm during the dissection, replace with fresh cold HBSS.
Note
Perform all following steps under a sterile tissue culture hood.
Once all cortices are collected, remove HBSS from 15 mL conical tube and add 4.5 mL warm (37 °C ) HBSS and 0.5 mL 2.5% trypsin.
After adding trypsin, invert the tube to mix.
Then incubate for 00:10:00 in a 37 °C water bath.
10m
Remove HBSS-trypsin solution with a 5 mL serological pipette.
Wash thrice with 7 mL attachment media.
Add attachment media, then wait until cortex tissue has settled at the bottom of the conical and remove attachment media with a serological pipette to repeat the washing step.
Note
We do not recommend using a vacuum aspirator for removing HBSS and attachment media, instead use a 10 mL serological pipette.
Add 5 mL attachment media after the last washing step.
Triturate cortices by pipetting up and down forcefully with a 5 mL serological pipette 10 – 15 times.
Note
Trituration is complete when no tissue clumps are visible and attachment media turns turbid.
Let media with triturated tissue settle for 00:01:00 - 00:02:00 .
3m
Transfer top 4.5 mL to a new tube to remove any remaining cell clumps.
Mix 10 µL cell suspension with 10 µL 0.5% trypan blue in an Eppendorf tube.
Count cells using a hemocytometer or an automated cell counter.
Dilute cortical neurons to 1,000,000 cells/mL.
For transfection and live-imaging, plate 200,000 cells per live-imaging dish.
Place imaging dishes in 37 °C cell culture incubator.
Note
Take up cells in a 200 µL pipette, then plate drops of cells in different areas to distribute neurons evenly across the live-imaging dish.
After 03:00:00 - 04:00:00 use an aspirator to remove all attachment media.
7h
Replace with 2 mL pre-equilibrated maintenance media per imaging dish.
Note
Cells should be attached to the glass-bottom dish at this point. Maintenance media must always be pre-equilibrated to 5% CO2 in 37 °C incubator before adding to cells.
Neuronal cell culture
Neuronal cell culture
On the day following the dissection, dilute AraC to 10 micromolar (µM) in maintenance media and bring to 37 °C .
Add 200 µL maintenance media + AraC to each imaging dish for a final AraC concentration of 1 micromolar (µM) .
Every 3-4 days, remove 600 µL maintenance media from each dish and replace with 750 µL fresh, pre-equilibrated maintenance media.
Note
Cultured neurons are sensitive. Try to keep time outside the cell culture incubator to a minimum. If possible, use a separate incubator for primary neurons and keep openings to a minimum.
Transfection
Transfection
16h 55m
16h 55m
Transfect primary neurons on DIV6-7, ~ 16:00:00 before live-imaging.
16h
Replace conditioned media with fresh, pre-equilibrated maintenance media (2 mL per imaging dish).
Save old media = conditioned media in a 10 cm cell culture dish at 37 °C in the cell culture incubator.
For each imaging dish, prepare two tubes with transfection reagents.
In tube 1, add plasmid DNA to 200 µL Neurobasal medium.
In tube 2, add Lipofectamine 2000 to 100 µL Neurobasal medium.
Note
It is important to use non-supplemented Neurobasal and not maintenance medium to set up the transfection reaction.
Note
The amount of Lipofectamine 2000 and plasmid DNA depends on the specific construct(s) used and may require optimization. We find that for transfection with one plasmid, 0.4 µg DNA and 1 µL Lipofectamine 2000 works well in most cases.
Combine contents of tube 1 + 2 and mix by gently pipetting up and down 4-5 times.
Incubate mix at Room temperature for 00:10:00 .
10m
Add Lipofectamine-DNA mix to imaging dishes dropwise.
Incubate for 00:45:00 at 37 °C in cell culture incubator.
45m
Remove all transfection media and replace with conditioned media collected earlier.
Return cells to incubator and image on the next day.