TITLE: Primary neuron culture for live imaging of axonal cargoes
AUTHORS: C. Alexander Boecker, Erika L.F. Holzbaur

[DESCRIPTION]
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. 

[STEPS]
SECTION: Day before dissection
1. Coat glass-bottom imaging dishes with PLL.
SECTION: Day before dissection
1.1.  Hydrate 100 mg PLL (Sigma) in 50 mL 0.1 Molarity (M) borate buffer, pH 8.5.

SECTION: Day before dissection
1.2. Store PLL stock solution (2 mg/mL) in 1 mL aliquots at -80 °C. 

SECTION: Day before dissection
1.3.  On the day before neuron dissection, dilute PLL in ddH2O 1:20 to a final concentration of 100 μg/ml. 

SECTION: Day before dissection
1.4.  Add 1 mL PLL to each glass-bottom imaging dish (MatTek) and incubate   at 37 °C. 

SECTION: Day before dissection
1.5. Only coat the glass center with PLL.
 

SECTION: Day before dissection
2. Prepare HBSS, attachment media and maintenance media.
SECTION: Day before dissection
2.1. For 500 mL 1x HBSS, combine

 
 

SECTION: Day before dissection
2.2.  Store 1x HBSS at 4 °C and use within one month. 

SECTION: Day before dissection
2.3.  For 50 mL attachment media, combine 

 

SECTION: Day before dissection
2.4. For 50 mL maintenance media, combine
 
 

SECTION: Dissection of cortical neurons
3. In the morning of the day of dissection, wash PLL-coated imaging dishes twice with sterile ddH2O.
SECTION: Dissection of cortical neurons
3.1. Add 2 mL attachment media per imaging dish and leave dishes at 37 °C in cell culture incubator. 

SECTION: Dissection of cortical neurons
3.2. Warm required amount of attachment media and 1x HBSS (4.5 mL for one dissection) in 37 °C water bath. 

SECTION: Dissection of cortical neurons
3.3.  Aliquot maintenance media into 10 cm cell culture dish to equilibrate in 37 °C / 5% CO2 cell culture incubator. 

SECTION: Dissection of cortical neurons
3.4.  Let 2.5% trypsin aliquots thaw at Room temperature. 

SECTION: Dissection of cortical neurons
4. Sacrifice pregnant mouse, dissect embryos, and place embryonic brains in HBSS on ice.

SECTION: Dissection of cortical neurons
4.1. Using a dissecting microscope, remove meninges from brain hemispheres with fine forceps.
SECTION: Dissection of cortical neurons
5. 
 

 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.

SECTION: Dissection of cortical neurons
4.2.  Isolate cortices using fine forceps and small spring scissors. 
SECTION: Dissection of cortical neurons
4.3.  Transfer dissected cortices into a 15 mL conical tube filled with 5 mL HBSS and keep on ice until all cortices are collected. 
 

 



SECTION: Dissection of cortical neurons
6. Remove HBSS-trypsin solution with a 5 mL serological pipette. 
SECTION: Dissection of cortical neurons
5.1. After adding trypsin, invert the tube to mix. 
SECTION: Dissection of cortical neurons
5.2. Then incubate for 10 min in a 37 °C water bath.

SECTION: Dissection of cortical neurons
6.1. Wash thrice with 7 mL attachment media.

SECTION: Dissection of cortical neurons
6.2. 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. 
 

SECTION: Dissection of cortical neurons
7. Add 5 mL attachment media after the last washing step.

SECTION: Dissection of cortical neurons
7.1. Triturate cortices by pipetting up and down forcefully with a 5 mL serological pipette 10 – 15 times. 
 

SECTION: Dissection of cortical neurons
7.2.  Let media with triturated tissue settle for 1 min- 2 min.

SECTION: Dissection of cortical neurons
7.3. Transfer top 4.5 mL to a new tube to remove any remaining cell clumps. 

SECTION: Dissection of cortical neurons
8. Mix 10 µL cell suspension with 10 µL 0.5% trypan blue in an Eppendorf tube.

SECTION: Dissection of cortical neurons
8.1.  Count cells using a hemocytometer or an automated cell counter.
SECTION: Dissection of cortical neurons
9. Dilute cortical neurons to 1,000,000 cells/mL. 
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9.1. For transfection and live-imaging, plate 200,000 cells per live-imaging dish. 
SECTION: Dissection of cortical neurons
9.2. Place imaging dishes in 37 °C cell culture incubator.
 


SECTION: Dissection of cortical neurons
10.       After 180 min- 240 min use an aspirator to remove all attachment media.


SECTION: Dissection of cortical neurons
10.1.  Replace with 2 mL pre-equilibrated maintenance media per imaging dish.
 


SECTION: Neuronal cell culture
11. On the day following the dissection, dilute AraC to 10 micromolar (µM) in maintenance media and bring to 37 °C.

SECTION: Neuronal cell culture
11.1. Add 200 µL maintenance media + AraC to each imaging dish for a final AraC concentration of 1 micromolar (µM).

SECTION: Neuronal cell culture
12. Every 3-4 days, remove 600 µL maintenance media from each dish and replace with 750 µL fresh, pre-equilibrated maintenance media. 
 



SECTION: Transfection
13. Transfect primary neurons on DIV6-7, ~ 960 min before live-imaging. 

SECTION: Transfection
14. Replace conditioned media with fresh, pre-equilibrated maintenance media (2 mL per imaging dish).

SECTION: Transfection
14.1.  Save old media = conditioned media in a 10 cm cell culture dish at 37 °C in the cell culture incubator. 

SECTION: Transfection
15.    For each imaging dish, prepare two tubes with transfection reagents.
SECTION: Transfection
15.1. In tube 1, add plasmid DNA to 200 µL Neurobasal medium. 

SECTION: Transfection
15.2.  In tube 2, add Lipofectamine 2000 to 100 µL Neurobasal medium. 
 

 


SECTION: Transfection
16. Combine contents of tube 1 + 2 and mix by gently pipetting up and down 4-5 times.
SECTION: Transfection
16.1.  Incubate mix at Room temperature for 10 min.

SECTION: Transfection
17.     Add Lipofectamine-DNA mix to imaging dishes dropwise.
SECTION: Transfection
17.1.  Incubate for 45 min at 37 °C in cell culture incubator. 

SECTION: Transfection
18. Remove all transfection media and replace with conditioned media collected earlier.
SECTION: Transfection
18.1.  Return cells to incubator and image on the next day. 
SECTION: Day before dissection
2.5. Store attachment media and maintenance media at 4 °C.