Jul 10, 2024
Preparing primary sandwich hippocampal neuron cultures for cryo-electron tomography
- Florelle Domart1,2,
- Arsen Petrovic1,3,
- Thanh Thao Do1,4,
- Anna Siegert1,4,
- Thomas Dresbach2,
- Rubén Fernández-Busnadiego1,2,3,4,5,6
- 1University Medical Center Göttingen, Institute for Neuropathology, Göttingen, 37077 Germany;
- 2University Medical Center Göttingen, Institute of Anatomy and Embryology, Göttingen, 37075 Germany;
- 3Collaborative Research Center 1286 "Quantitative Synaptology", University of Göttingen, Göttingen, Germany;
- 4Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, USA;
- 5Cluster of Excellence “Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells” (MBExC), University of Göttingen, Göttingen, 37077, Germany;
- 6Faculty of Physics, University of Göttingen, Göttingen, 37077, Germany
Protocol Citation: Florelle Domart, Arsen Petrovic, Thanh Thao Do, Anna Siegert, Thomas Dresbach, Rubén Fernández-Busnadiego 2024. Preparing primary sandwich hippocampal neuron cultures for cryo-electron tomography. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr3y83vmk/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: December 27, 2023
Last Modified: July 10, 2024
Protocol Integer ID: 92910
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-000282
Deutsche Forschungsgemeinschaft
Grant ID: EXC 2067/1-390729940
Deutsche Forschungsgemeinschaft
Grant ID: 448415290
Deutsche Forschungsgemeinschaft
Grant ID: SFB1286/A12
Deutsche Forschungsgemeinschaft/NeuroNex
Grant ID: FE 1940
Abstract
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.
Attachments
pd2vbiezx.pdf
292KB
Guidelines
Materials
Astrocyte feeder layer
- T75 flasks
- 10 mL serological pipettes
- 15 mL sterile tubes
- 25 mm sterile coverslips
| A | B | |
| MEM 1X (Gibco) supplemented with | ||
| L-Glutamine | 2 mM | |
| Horse Serum (Gibco) | 10 % | |
| Glucose (MEM10%HS) | 4.65 g/L | |
- 0.1 mg/mL poly-L-lysine;
- Sterile water;
- 0.05 % Trypsin/0.02 % EDTA
- DMSO
- Centrifuge for 15 mL tubes.
Primary neuron cultures with astrocyte feeder layer
- Sterile tweezers
| A | B | |
| Neurobasal medium supplemented with | ||
| B27 | 2 % | |
| L-Glutamine | 2 mM | |
- DMEM10%FCS
- Arabinofuranoside (cytosine-β-arabinofuranoside hydrochloride)
Preparation of the EM grids
Preparation of the EM grids
For preparation of EM grids, follow the accompanying protocol by Siegert, Petrovic, Do et al.
Note
Only 35 mm dishes with 4 inner rings are suitable for the preparation of primary sandwich
hippocampal neuron cultures.
Preparation of primary sandwich hippocampal neuron culture on EM grids - Astrocyte feeder layer
Preparation of primary sandwich hippocampal neuron culture on EM grids - Astrocyte feeder layer
Plate 5 million cortical cells from an E19 rat embryo cortical suspension in T75 flask in MEM10%HS.
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.
Note
The choice of a specific medium as well as the removal of microglia (as described in the
previous step) favours the proliferation of astrocytes over neurons and other glia in the cortical cell suspension.
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 00:15:00 at Room temperature .
Fig.1: Timeline of primary sandwich hippocampal neuron culture preparation. Astrocytes are cultured for 3 weeks prior to plating on coverslips. Following the hippocampal dissection, primary hippocampal cultures are plated on EM grids, and neuronal maturation takes place in the presence of the astrocyte coverslips and arabinofuranoside.
15m
Wash the coverslips 3 times with sterile water and replace the water with MEM10%HS.
Harvest the astrocytes from the flask: Slam the flask and aspirate the medium.
Quickly wash the flask with 0.05 % Trypsin/0.02 % EDTA.
Add 2 mL of trypsin/EDTA and incubate 00:02:00 at 37 °C until the cells detach.
2m
Stop the trypsination by adding 5 mL of MEM10%HS.
Release the cells by multiple rounds of pipetting with a 10 mL serological pipette.
Transfer the cells to a 15 mL sterile tube and centrifuge for 00:05:00 at 500 x g .
5m
Resuspend the cells in 2 mL of MEM10%HS complete medium.
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.
Note
The remaining astrocytes in MEM10%HS complete medium with 10 % DMSO can be frozen and kept in liquid nitrogen storage for more than a year. They can be thawed six days before the hippocampal dissection.
One day after plating, change the medium of the astrocytes again with fresh preequilibrated MEM10%HS.
Preparation of primary sandwich hippocampal neuron culture on EM grids - Primary neuron cultures with astrocyte feeder layer
Preparation of primary sandwich hippocampal neuron culture on EM grids - Primary neuron cultures with astrocyte feeder layer
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.
Dilute the hippocampal suspension to reach a density of 200,000 to 350,000 cells per mL in DMEM10%FCS.
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.
Fig 2: Schema of the sandwich cultures on EM grids
After two hours, add 500 µL of pre-heated DMEM10%FCS per dish.
Incubate Overnight at 37 °C and 5 % CO2.
Replace the plating medium with 2 mL of pre-conditioned Neurobasal medium from the astrocyte feeder layers’ dish.
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).
Fig 2: Schema of the sandwich cultures on EM grids
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.
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.
The neurons are considered mature starting from 15 days in vitro (DIV15).
Protocol references
Kaech, S., and Banker, G. (2006). Culturing hippocampal neurons. Nature Protocols 1, 2406- 2415.