TITLE: Preparing primary sandwich hippocampal neuron cultures for cryo-electron tomography
AUTHORS: Florelle Domart, Arsen Petrovic, Thanh Thao Do, Anna Siegert, Thomas Dresbach, Rubén Fernández-Busnadiego

[DESCRIPTION]

Primary sandwich hippocampal neuron cultures are adapted from the Kaech and Banker protocol (Kaech and Banker, 2006) and provide neuronal cultures with almost no glial cells, which may facilitate the targeting of neurons for cryo-electron tomography (see accompanying protocol by Siegert, Petrovic, Do et al.). In addition, the cells can be seeded at a very low density.




[STEPS]
SECTION: Preparation of the EM grids
1. For preparation of EM grids, follow the accompanying protocol by Siegert, Petrovic, Do et al.
 

SECTION: Preparation of primary sandwich hippocampal neuron culture on EM grids - Astrocyte feeder layer
2. Plate 5 million cortical cells from an E19 rat embryo cortical suspension in T75 flask in MEM10%HS.
SECTION: Preparation of primary sandwich hippocampal neuron culture on EM grids - Astrocyte feeder layer
3. Change the medium of the astrocytes twice a week with fresh MEM10%HS preequilibrated at 37 °C and 5 % CO2. Slam the flask against the bench surface before aspirating the medium to remove the microglial cells. Two weeks after the dissection, the astrocyte cultures should be more than 50 % confluent.

 

SECTION: Preparation of primary sandwich hippocampal neuron culture on EM grids - Astrocyte feeder layer
4. 6-7 days before the hippocampal dissection (2 weeks after the cortical dissection; Fig.1), coat 25 mm sterile coverslips with 0.1 mg/mL poly-L-lysine for 15 min at Room temperature. 


 

SECTION: Preparation of primary sandwich hippocampal neuron culture on EM grids - Astrocyte feeder layer
5. Harvest the astrocytes from the flask: Slam the flask and aspirate the medium. 
SECTION: Preparation of primary sandwich hippocampal neuron culture on EM grids - Astrocyte feeder layer
4.1. Wash the coverslips 3 times with sterile water and replace the water with MEM10%HS.
SECTION: Preparation of primary sandwich hippocampal neuron culture on EM grids - Astrocyte feeder layer
5.1. Quickly wash the flask with 0.05 % Trypsin/0.02 % EDTA.
SECTION: Preparation of primary sandwich hippocampal neuron culture on EM grids - Astrocyte feeder layer
5.2. Add 2 mL of trypsin/EDTA and incubate 2 min at 37 °C until the cells detach. 

SECTION: Preparation of primary sandwich hippocampal neuron culture on EM grids - Astrocyte feeder layer
5.3. Stop the trypsination by adding 5 mL of MEM10%HS.

SECTION: Preparation of primary sandwich hippocampal neuron culture on EM grids - Astrocyte feeder layer
5.4. Release the cells by multiple rounds of pipetting with a 10 mL serological pipette. 
SECTION: Preparation of primary sandwich hippocampal neuron culture on EM grids - Astrocyte feeder layer
5.5. Transfer the cells to a 15 mL sterile tube and centrifuge for 5 min at 500 x g.

SECTION: Preparation of primary sandwich hippocampal neuron culture on EM grids - Astrocyte feeder layer
5.6.  Resuspend the cells in 2 mL of MEM10%HS complete medium. 

SECTION: Preparation of primary sandwich hippocampal neuron culture on EM grids - Astrocyte feeder layer
5.7. Count the cells and plate them dropwise on each of the coverslips: use 200,000 cells in a total volume of 100 µL of medium per coverslip.

 

SECTION: Preparation of primary sandwich hippocampal neuron culture on EM grids - Astrocyte feeder layer
6. One day after plating, change the medium of the astrocytes again with fresh preequilibrated MEM10%HS.
SECTION: Preparation of primary sandwich hippocampal neuron culture on EM grids - Primary neuron cultures with astrocyte feeder layer
7. One day before the hippocampal dissection, precondition the astrocyte feeder layer in Neurobasal medium + 2 % B27 + 2 millimolar (mM) L-Glutamine pre-incubated at 37 °C and 5 % CO2 for few hours.

SECTION: Preparation of primary sandwich hippocampal neuron culture on EM grids - Primary neuron cultures with astrocyte feeder layer
8. Dilute the hippocampal suspension to reach a density of 200,000 to 350,000 cells per mL in DMEM10%FCS.
SECTION: Preparation of primary sandwich hippocampal neuron culture on EM grids - Primary neuron cultures with astrocyte feeder layer
9. Immediately after removing the water from the compartments of the dish plate 100 µL of cell suspension dropwise on each EM grid (Fig. 2, left). Incubate at 37 °C and 5 % CO2.

 


SECTION: Preparation of primary sandwich hippocampal neuron culture on EM grids - Primary neuron cultures with astrocyte feeder layer
10. After two hours, add 500 µL of pre-heated DMEM10%FCS per dish.

SECTION: Preparation of primary sandwich hippocampal neuron culture on EM grids - Primary neuron cultures with astrocyte feeder layer
11. Incubate   at 37 °C and 5 % CO2.

SECTION: Preparation of primary sandwich hippocampal neuron culture on EM grids - Primary neuron cultures with astrocyte feeder layer
12. Replace the plating medium with 2 mL of pre-conditioned Neurobasal medium from the astrocyte feeder layers’ dish.

SECTION: Preparation of primary sandwich hippocampal neuron culture on EM grids - Primary neuron cultures with astrocyte feeder layer
13. With sterile tweezers take the coverslip with the astrocyte layer and flip the coverslip so that the neurons are facing the astrocytes (Fig. 2, right). 

 


SECTION: Preparation of primary sandwich hippocampal neuron culture on EM grids - Primary neuron cultures with astrocyte feeder layer
14. To stop the proliferation of glial cells on the grids, treat the primary neuron cultures with the anti mitotic agent arabinofuranoside (cytosine-β-arabinofuranoside hydrochloride) three days after plating the hippocampal cells. 
SECTION: Preparation of primary sandwich hippocampal neuron culture on EM grids - Primary neuron cultures with astrocyte feeder layer
15. The neurons are considered mature starting from 15 days in vitro (DIV15).
SECTION: Preparation of primary sandwich hippocampal neuron culture on EM grids - Primary neuron cultures with astrocyte feeder layer
14.1. Add arabinofuranoside to a final concentration of 2.45 micromolar (µM) to each dish and distribute evenly by gently moving the dish in a circular motion.