Primary neuron culture for live imaging of axonal cargoes
C. Alexander Boecker, Erika L.F. Holzbaur
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
Steps
Day before dissection
Coat glass-bottom imaging dishes with PLL.
Hydrate 100mg PLL (Sigma) in 50mL 0.1Molarity (M) borate buffer, 8.5.
Store PLL stock solution (2mg/mL) in 1mL aliquots at -80°C.
On the day before neuron dissection, dilute PLL in ddH2O 1:20 to a final concentration of 100μg/ml.
Add 1mL PLL to each glass-bottom imaging dish (MatTek) and incubate at 37°C.
Only coat the glass center with PLL.
Prepare HBSS, attachment media and maintenance media.
For 500mL 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 50mL 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 50mL 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.
Dissection of cortical neurons
In the morning of the day of dissection, wash PLL-coated imaging dishes twice with sterile ddH2O.
Add 2mL attachment media per imaging dish and leave dishes at 37°C in cell culture incubator.
Warm required amount of attachment media and 1x HBSS (4.5mL 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 5mL HBSS and keep On ice until all cortices are collected.
Once all cortices are collected, remove HBSS from 15 mL conical tube and add 4.5mL warm (37°C) HBSS and 0.5mL 2.5% trypsin.
After adding trypsin, invert the tube to mix.
Then incubate for 0h 10m 0s in a 37°C water bath.
Remove HBSS-trypsin solution with a 5 mL serological pipette.
Wash thrice with 7mL 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.
Add 5mL attachment media after the last washing step.
Triturate cortices by pipetting up and down forcefully with a 5 mL serological pipette 10 – 15 times.
Let media with triturated tissue settle for 0h 1m 0s- 0h 2m 0s.
Transfer top 4.5mL to a new tube to remove any remaining cell clumps.
Mix 10µL cell suspension with 10µL 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.
After `3h 0m 0s`- `4h 0m 0s` use an aspirator to remove all attachment media.
Replace with 2mL pre-equilibrated maintenance media per imaging dish.
Neuronal cell culture
On the day following the dissection, dilute AraC to 10micromolar (µM) in maintenance media and bring to 37°C.
Add 200µL maintenance media + AraC to each imaging dish for a final AraC concentration of 1micromolar (µM).
Every 3-4 days, remove 600µL maintenance media from each dish and replace with 750µL fresh, pre-equilibrated maintenance media.
Transfection
Transfect primary neurons on DIV6-7, ~ 16h 0m 0s before live-imaging.
Replace conditioned media with fresh, pre-equilibrated maintenance media (2mL 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 200µL Neurobasal medium.
In tube 2, add Lipofectamine 2000 to 100µL Neurobasal medium.
Combine contents of tube 1 + 2 and mix by gently pipetting up and down 4-5 times.
Incubate mix at Room temperature for 0h 10m 0s.
Add Lipofectamine-DNA mix to imaging dishes dropwise.
Incubate for 0h 45m 0s at 37°C in cell culture incubator.
Remove all transfection media and replace with conditioned media collected earlier.
Return cells to incubator and image on the next day.
