A Scalable Method to Study Neuronal Survival in Primary Neuronal Culture with Single-cell and Real-Time Resolution
Summary
Here, we present a simple, potent, and versatile methodology to investigate neuronal survival upon cytotoxic stress in primary cortical neurons with cellular resolution in real time or in fixed material.
Abstract
Neuronal loss is at the core of many neuropathologies, including stroke, Alzheimer’s disease, and Parkinson’s disease. Different methods were developed to study the process of neuronal survival upon cytotoxic stress. Most methods are based on biochemical approaches that do not allow single-cell resolution or involve complex and costly methodologies. Presented here is a versatile, inexpensive, and effective experimental paradigm to study neuronal survival. This method takes advantage of sparse fluorescent labeling of the neurons followed by live imaging and automated quantification. To this aim, the neurons are electroporated to express fluorescent markers and co-cultured with non-electroporated neurons to easily regulate cell density and increase survival.
Sparse labeling by electroporation allows a simple and robust automated quantification. In addition, fluorescent labeling can be combined with the co-expression of a gene of interest to study specific molecular pathways. Here, we present a model of stroke as a neurotoxic model, namely, the oxygen-glucose deprivation (OGD) assay, which was performed in an affordable and robust homemade hypoxic chamber. Finally, two different workflows are described using IN Cell Analyzer 2200 or the open-source ImageJ for image analysis for semi-automatic data processing. This workflow can be easily adapted to different experimental models of toxicity and scaled up for high-throughput screening. In conclusion, the described protocol provides an approachable, affordable, and effective in vitro model of neurotoxicity, which can be suitable for testing the roles of specific genes and pathways in live imaging and for high-throughput drug screening.
Introduction
The study of neuronal disorders requires experimental models that are amenable to genetic, molecular, and cellular analyses. Primary cortical neurons are a very potent model for studying neuronal survival and toxicity1,2,3,4. Under the appropriate conditions, primary neurons will progressively develop synaptic contacts and present hallmarks of mature neurons. Therefore, this model is more reliable than immortalized cell lines in modeling the physiology of the neurons and more amenable to manipulations than animal models. However, in comparison to cell lines, primary cortical neurons are difficult to transfect and relatively fragile. Moreover, the intricate morphology of cortical neurons may complicate the imaging and analysis in high-density cultures. Conversely, low-density primary neuronal cultures are easier to analyze but tend to be more fragile.
Taking all of this into account, this paper presents an affordable, versatile, and scalable in vitro workflow to model and investigate neuronal survival (Figure 1). Key points of this protocol are i) the method of in vitro electroporation of the neurons with a fluorescent marker to allow sparse labeling and imaging of live cells; ii) its versatility in different models of cytotoxicity; and iii) the semi-automated or automated analysis.
The electroporation system (see the Table of Materials) provides an open and inexpensive procedure that does not involve kits and specific solutions4,5,6. Moreover, this method can be easily adapted to obtain an optimal transformation efficiency and density of transfected neurons, mixing electroporated with non-electroporated cells. The co-culture with naïve (non-electroporated) neurons significantly improves the viability of the electroporated cells compared to low-density cultures. Moreover, it enables sparse labeling with an adjustable density of the electroporated neurons, maintaining a consistent level of gene expression. It is important to aim for 3-5% of electroporated neurons in survival assays.
Having sparse labeling essentially facilitates the automation of the image analysis because the cells are well-separated and easily distinguishable. Notably, this experimental paradigm may be adapted to test multiple genes of interest by simultaneously co-culturing neurons co-electroporated with different cytosolic fluorescent markers (e.g., cyan fluorescent protein, red fluorescent protein (RFP), green fluorescent protein (GFP)) in the same well. Similar to other cytotoxicity assays (e.g., propidium iodide or lactate dehydrogenase), the assay described herein is based on the fact that neuronal death is accompanied by rupturing of the cell membrane. This provokes the release of cytosolic proteins by diffusion and, consequently, the loss of GFP fluorescence.
An in vitro model of stroke, namely the OGD model, is presented as an example of neurotoxicity7,8,9. This protocol entails exposing the primary neurons to a salt buffer similar to an artificial cerebrospinal fluid but deprived of oxygen and glucose. Although this model has been presented as an example of neurotoxic stress, different cytotoxic conditions can be tested with the same workflow8,9. Finally, sparse labeling easily enables the development of automated imaging analysis. Here, a protocol was established based on standard immunofluorescence and ImageJ analysis in a smaller setup. Next, this workflow was adapted and scaled up using a cell imaging system that allows an automatized analysis in live imaging in mid- and high-throughput modes. In conclusion, this paper presents a flexible, affordable, and scalable methodology to study neuronal survival in different experimental models of toxicity using live imaging and automated quantification.
Protocol
Representative Results
Discussion
This protocol shows an effective way of modeling a stroke in vitro. To achieve this goal, we proposed sparse labeling of cortical neurons using the electroporation system NEPA215. This is an open system that allows customization of the protocol with minimal operative cost compared to other systems that employ kits or specific devices. Mixed culture of naïve and electroporated neurons allows more flexibility and robustness as compared to low-density neuronal culture. This allows the s…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We would like to thank Carlos Dotti for sharing his expertise in neuronal culture. We also thank Alicia Martínez-Romero from the Cytomics Core Facility of the Centro de Investigación Principe Felipe (CIPF), which is supported by European FEDERER funding. The project is supported by the Spanish Ministry of Economy and Competitiveness for (SAF2017-89020-R) reagents, materials, and the salaries of YDC and PF. PF is also supported by the grant RyC-2014-16410. CGN and PF are supported by Conselleria de Sanitat of the Generalitat Valenciana, as well as AGM (ACIF/2019/015). Ángela Rodríguez Prieto is supported by the Spanish Ministry of Science, Innovation and Universities, with the grant PRE2018-083562.
Materials
| 10 cm petri dishes | FISHER SCIENTIFIC, S.L. | 1130-9283 | |
| 3.5 cm petri dishes | Sterilin | ||
| 75 cm2 flask | Corning | 430641U | |
| B-27 | Life Technologies | 17504-044 | |
| Cell culture plates | Corning incorporation Costar® | 3513 | |
| Cell incubator | Thermo Electron Corporation | Model 371 | |
| Cell strainer 70 µm | Falcon | 352350 | |
| Cold lights | Schott | 223488 | KL 1500 LCD |
| Coverslips (15 mm) | Marienfeld | 111530 | |
| CU500 Cuvette Chamber | Nepa Gene | ||
| CU600 Cuvette Stand Holder | Nepa Gene | ||
| DAPI | Sigma-Aldrich Quimica, S. L. | D9542-10MG | 1:2000 |
| DMSO | Panreac | A3672 | |
| Dumont Fine Forceps | FST | 11254-20 | |
| Dumont Fine Forceps | FST | 11252-00 | |
| EC-002S NEPA Electroporation Cuvettes, 2mm gap | Nepa Gene | ||
| Filter strainer | Falcon | 352350 | |
| Fine Scissors-Sharp-Blunt | FST | 14028-10 | |
| Fine Scissors-ToughCut r | FST | 14058-09 | |
| GFP chicken IgY | Aves Labs | GFP-1010 | 1:600 |
| Glucose | Sigma | 68769-100ml | |
| GlutaMAX-I Supplement 200 mM 100 mL | Fisher Scientific | 35050-061 | |
| HBSS | Thermofisher | 14175-095 | https://www.thermofisher.com/es/es/home/technical-resources/media-formulation.156.html |
| Hepes 1 M | ThermoFisher | 15630-080 | |
| Horse Serum | Invitrogen | 26050088 | |
| MEM | Thermofisher | 11095080 | https://www.thermofisher.com/order/catalog/product/11095080#/11095080 |
| Microscope slide (polilysine) | VWR | 631-0107 | Dimension: 25 x 75 x 1 mm |
| Mowiol 4-88 | Sigma-Aldrich Quimica, S. L. | 81381-250G | |
| Needles yellow, 30 gauge | BD Microlance™ 3 | 304000 | |
| NEPA21 electroporator | Nepa Gene | ||
| Neubauer chamber | Blau Brand | 717805 | |
| Neurobasal Medium | ThermoFisher | 21103-49 | |
| Opti-MEM | Invitrogen | 31985-062 | |
| Parafilm | Cole-Parmer | PM996 | |
| Paraformaldehyde (PFA), 95% | Sigma-Aldrich Quimica, S. L. | 158127-500G | Use solution: 4% |
| PEI | Polysciences | 23966-1 | |
| Plasmid for GFP | pCMV-GFP-ires-Cre, described in Fazzari et al., Nature, 2010 | ||
| Poly-L-Lysine | SIGMA | P2636 | |
| PS ( Penicillin, Streptomycin) | ThermoFisher | 15140122 | |
| Serrate forceps | FST | 11152-10 | |
| Stereomicroscope | WORLD PRECISION INSTRUMENTS | ||
| Syringes | BD Plastipak 1ml | 303176 | |
| Triton X-100 | Sigma-Aldrich Quimica, S. L. | MDL number: MFCD00128254 | Non-ionic |
| Trypsin-EDTA | ThermoFisher | 25300054 | |
| Tubes 15 mL | Fisher | 05-539-4 | |
| Tubes 50 mL | VWR | 21008-242 | |
| Tupperware | – | – | Hermetic tupperware with screw lid. SP Berner – Taper 1 L Redondo con Rosca. Any equivalent hermetic Tupperware may be purchased in any supermarket. |
| Water bath | SHELDON LABORATORY MODEL 1224 | 1641951 | |
Riferimenti
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