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 toxicity^1,2,3,4. 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 comparis....

Protocol

All procedures using animals should be supervised by the bioethical animal committee of the institute and performed in compliance with local regulations. The procedures presented herein were approved by the delegated authority and comply with the regulations in Spain and Europe.

1. Primary neuronal culture

NOTE: All the steps are performed inside the culture hood, using sterile materials and solutions to maintain sterile conditions.

1. Poly-L-lysine (PLL) coating
   1. Wash the coverslips (CVs, 15 mm) in 70% ethanol overnight (o/n) in an orbital shaker.
      NOTE: The speed of the orbital shaker....

Results

This protocol aims to establish an in vitro model of stroke. It is important to obtain an adequate neuronal density, which will allow the recognition of individual electroporated neurons to analyze them individually. The stage of the neuronal culture after plating is also crucial. The maturation of neurons in culture is progressive. The dependence on growth factors, neurite outgrowth, connectivity, and electrophysiological activity will vary greatly depending on the stage. In these specific conditions at 4-6 day.......

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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