Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Jian Park; Ruoqi Yu; Yifei Dong

doi:10.3791/67078

JoVE Journal > Neuroscience

Neurociência

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Published: July 26, 2024

doi:

10.3791/67078

Jian Park*¹, Ruoqi Yu*¹, Yifei Dong¹

¹Department of Biochemistry, Microbiology & Immunology,University of Saskatchewan

Summary

This protocol describes a microglia-neuronal co-culture established from primary neuronal cells isolated from mouse embryos at embryonic days 15-16 and primary microglia generated from the brains of neonatal mice at post-natal days 1-2.

Abstract

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

Introduction

Microglia are tissue-resident macrophages that facilitate immunosurveillance and homeostasis in the central nervous system (CNS)¹^,²^,³. They originate from yolk sac erythromyeloid progenitor cells that colonize the brain during embryonic development⁴^,⁵^,⁶ and are maintained throughout the organism's life span through self-renewal, which involves proliferation and apoptosis⁷. At steady-state, resting microglia have ramified morphology and engage in tissue surveillance⁸^,⁹^,¹⁰.

Microglia express numerous cell-surface receptors, which enables them to rapidly respond to changes in the CNS¹¹^,¹² and to promote inflammatory responses in the event of infections or tissue injury¹²^,¹³^,¹⁴, as well as during neurodegenerative diseases⁹^,¹⁵, such as multiple sclerosis (MS)¹⁶^,¹⁷. Microglia also express receptors to various neurotransmitters and neuropeptides¹⁸^,¹⁹^,²⁰, which suggests they may also respond to and regulate neuronal activity²¹^,²². Indeed, microglia and neurons interact in various forms of bidirectional communication⁸^,²³ such as direct interactions mediated by membrane proteins or indirect interactions through soluble factors or intermediate cells²³^,²⁴.

For instance, various neurotransmitters secreted by neurons can modulate the neuroprotective or inflammatory activity of microglia²⁵^,²⁶^,²⁷. Additionally, direct interactions between neurons and microglia help to maintain microglia in a homeostatic state²⁸. Conversely, direct interactions of microglia with neurons can shape neuronal circuitry²⁹ and influence neuronal signaling³⁰^,³¹^,³². As disruptions of these interactions induce hyperexcitability of neurons³⁰ and microglia reactivity³³^,³⁴, dysregulated microglia-neuronal interactions are implicated as a contributing factor to neurological diseases³³^,³⁵. Indeed, psychotic²³^,²⁶ and neurodegenerative diseases have been described to exhibit dysfunctional microglia-neuronal interactions³³. While these observations highlight the importance of microglia-neuronal communication in the CNS, specific mechanisms of how these interactions regulate microglial and neuronal functions in health and disease are relatively unknown.

Within a complex milieu such as the CNS, multiple environmental factors can influence microglia-neuronal interactions, which limits the ability to study transient cellular interactions in vivo. Here, we present an in vitro microglia-neuronal co-culture system that can be used to study direct cellular interactions between microglia and neurons. This protocol describes the generation of primary microglia and neurons from the cortices of neonatal mice between post-natal days 0-2 and embryonic mice days 16-18, respectively. Neurons and microglia are then co-cultured in 96-well plates for downstream high-throughput experiments. We previously used this approach to demonstrate that microglia phagocytosis protects neurons from oxidized phosphatidylcholine mediated cell death³⁷, suggesting that this method can help to understand the roles of microglia in the context of neurodegeneration and MS. Similarly, microglia-neuronal co-cultures may also be useful for investigating the impact of microglia-neuronal crosstalk in other contexts such as viral infections³⁸ or neuronal injury and repair³⁹. Overall, in vitro microglia-neuronal co-culture systems enable researchers to study microglia-neuronal interactions in a manipulatable and controlled environment, which complements in vivo models.

Protocol

All animals used in this study were housed and handled with approval from the University Animal Care Committee (UACC) of the University of Saskatchewan and the Canadian Council on Animal Care (CCAC). Post-natal days 0-2 CD1 male and female mice and embryonic days 16-18 (E16-18) embryos from pregnant CD1 mice were used for this study. The details of the reagents and the equipment used are listed in the Table of Materials.

1. Primary microglia culture

NOTE: It is crucial to time the mixed glia and neuronal cultures so that microglia are mature and ready for harvest within 2 days after neurons are seeded into a 96-well plate.

Preparation
1. Pre-warm complete glia culture media, which includes Dulbecco's Modified Eagle Medium (DMEM) with high glucose supplemented with 10% heat-inactivated fetal bovine serum, 50 U/mL Penicillin/Streptomycin with 2.92 mg/mL of L-glutamine, 1mM sodium pyruvate, 1x non-essential amino acids, and 20 mM of HEPES in a 37 °C water bath.
2. Add 5 mL of 10 µg/mL poly-L-ornithine (PO) hydrobromide to each T-75 flask. Gently swirl the flask to ensure that PO completely covers the bottom of the flask. Incubate at 37 °C in a 5% CO₂ incubator for at least 1 h.
  NOTE: This coating step is required to facilitate cell adhesion to the T-75 flasks.
Isolation of brains from P0-P2 neonatal mice
NOTE: Sterilize all dissection tools and maintain the sterility of reagents. Ensure adherence to aseptic techniques throughout.
1. Before starting dissection, spray down the countertop with 70% ethanol or disinfecting spray where dissection will take place. Set up the dissection microscope and place the Petri dish under the microscope. Pour 20 mL of Leibovitz's L-15 media into the petri dish. Keep the lid of the Petri dish on while the microscope is not in use to reduce contamination.
2. Place down several layers of paper towel and thoroughly spray the paper towels with 70% ethanol. Prepare the dissection tools (dissection scissors, a pair of micro-forceps, and tissue forceps) by thoroughly spraying them with 70% ethanol and laying the tools down on the paper towels.
3. Transfer post-natal days 0-2 mouse onto the paper towels and thoroughly spray the mouse with 70% ethanol. Decapitate the mouse using dissection scissors (following institutionally approved protocols).
4. While gently holding the head, create a midline incision in the cranium starting from the neck to the nose. Peel the skull back with tissue forceps to reveal the brain.
5. Using tissue forceps, gently scoop the brain out by inserting the forceps under the brain and lift the brain out from the head. Immediately place the brain into the Petri dish containing 20 mL of Leibovitz's L-15 media.
6. Under a dissection microscope, carefully remove the brain stem and the meninges using a pair of micro-forceps. Collect the brains in a 50 mL conical tube containing 5 mL of Leibovitz's L-15 media.
Brain dissociation, seeding and maintaining mixed glia culture
NOTE: Perform all subsequent steps in a biosafety cabinet.
1. Mince the brains into approximately 1 mm² pieces with a sterile disposable scalpel blade. Carefully transfer the minced brains into a new sterile 50 mL tube.
2. To the minced brains, add a final concentration of 0.25% trypsin and incubate the tissue in a 37 °C water bath for 20 min. Every 2-3 min, mix the solution by gently inverting the tube to aid tissue dissociation.
3. From the 5% CO₂ incubator, transfer the T-75 flasks containing 5 mL of PO into the biosafety cabinet. Discard the PO and rinse the flasks 3 times with sterile 1x phosphate buffer solution (PBS). Ensure to thoroughly rinse the flasks as excess PO is toxic to cells.
4. Transfer the digested tissue from the 37 °C water bath into the biosafety cabinet. Add 5 mL of complete glia culture media to neutralize the trypsin. Gently mix the tissue suspension using a 10 mL pipette.
5. Place a sterile 70 µm nylon mesh cell strainer onto a sterile 50 mL conical tube and wet the mesh by pouring roughly 5 mL of L-15. Carefully decant the cell suspension through the cell strainer. Remove the plunger from a sterile 1 mL syringe, grind the tissue into the mesh using the plunger to get rid of tissue clumps, and generate a homogenized cell suspension.
6. Periodically, pour approximately 5 mL of Leibovitz's L-15 media into the 70 µm nylon mesh and continue grinding the tissue on the mesh with the 1 mL syringe plunger. When little to no visible tissue remains on the mesh, discard the mesh and set the cell suspension aside.
7. Mix the cell suspension by gently pipetting up and down. To each flask, add equal volumes of cell suspension containing 1-2 brains with glia culture media to a final volume of 15 mL.
8. Incubate the cells at 37 °C in a 5% CO₂ incubator overnight. The following day, wash the flasks twice with sterile 1x PBS to remove unattached cells and debris.
9. Add 15 mL of fresh complete glia culture media to the culture and incubate at 37 °C in a 5% CO₂ incubator. After 3 days, change half the media with 10 mL of fresh glia culture media supplemented with 40 ng/mL of macrophage colony-stimulating factor (MCSF).
  NOTE: Thereafter, half the media is replaced with 10 mL of fresh glia culture media supplemented with 40 ng/mL of MCSF every 3 days. The culture is maintained for 8-10 days before microglia are harvested.

2. Primary neuron culture

Preparation
1. Thaw B-27 plus supplement from B-27 plus neuronal culture system. Pre-warm neurobasal plus medium from B-27 plus neuronal culture system, 1x Hank's Balanced Salt Solution (HBSS), and heat-inactivated fetal bovine serum (FBS) at 37 °C in a water bath.
2. Coat T-25 flasks with 5 mL of 10 µg/mL PO and incubate the flasks for at least 1 hour at 37 °C. Prepare instruments and supplies for dissection.
  NOTE: This coating step facilitates cell adhesion to the T-25 flasks.
Brain isolation from E16-E18 mouse embryos
NOTE: Sterilize all dissection tools and maintain the sterility of reagents. Ensure adherence to aseptic techniques throughout.
1. Before starting dissection, spray down the countertop with 70% ethanol or disinfecting spray where dissection will take place. Set up the dissection microscope and place the Petri dish under the microscope. Pour 20 mL of 1x HBSS into the Petri dish. Keep the lid of the Petri dish on while the microscope is not in use to reduce contamination.
2. Place down several layers of paper towel and thoroughly spray the paper towels with 70% ethanol. Prepare the dissection tools (spring scissors, a pair of micro-forceps, and tissue forceps) by thoroughly spraying them with 70% ethanol and laying the tools down on the paper towels.
3. Anesthetize a pregnant mouse between gestation days 16-18 using isoflurane and euthanize the mouse by cervical dislocation (following institutionally approved protocols).
4. Lay the mouse on its back on a piece of paper towel soaked in 70% ethanol. Disinfect the abdominal area with 70% ethanol. Lift the skin of the lower abdomen with tissue forceps and perform a V-shaped incision from this point to the lower rib cage with dissection scissors.
5. Grab the uterine horns containing the embryos with tissue forceps and gently remove the embryos from the abdominal cavity. Briefly disinfect the uterine horns containing embryos with 70% ethanol.
6. Dissect one embryo from its individual sac at a time. Using spring scissors, create a midline incision at the top of the skull, starting from the back of the neck to the nose. Then, lift the skull away from the incision site to reveal the brain. Gently scoop out the brain with tissue forceps and transfer the brain to a 10 cm Petri dish containing 15 mL of 1x HBSS.
7. Under a dissection microscope, carefully remove the brainstem and the meninges on both sides of the brain cortices with a pair of micro-forceps. Transfer the brain cortices into a 50 mL conical tube containing 10 mL of 1x HBSS and place on ice.
Brain dissociation and seeding of cortical neurons in cell culture
NOTE: Perform all subsequent steps in a biosafety cabinet.
1. Mince the brains into approximately 1 mm² pieces in the 50 mL conical tube with a sterile disposable scalpel blade. Transfer the minced brains into a new sterile 50 mL conical tube.
2. Add trypsin to the tube with a final concentration of 0.25%. Immerse the tube in a 35-38 °C hot water bath for 15 min and gently mix the tube to homogenize the tissue every 2-3 min.
3. Take the tube out of the hot water bath. Gently pipette the tissue suspension up and down to further dissociate cells, then add 0.5 mL of heat-inactivated FBS to the homogenized suspension to neutralize trypsin activity. Avoid air bubbles to minimize toxicity to neuronal cells.
4. Place a sterile 70 µm nylon mesh cell strainer onto a sterile 50 mL conical tube and wet the mesh by adding roughly 5 mL of neurobasal media. Filter the cell suspension through the 70 µm nylon mesh cell strainer. Using a 1 mL syringe plunger, gently grind the tissue into the cell strainer. Periodically pour approximately 5 mL of 1x HBSS while continuing grinding to wash the cell strainer.
5. Centrifuge the conical tube at 300 x g for 5 min at 4 °C. During this time, dilute B-27 plus supplement in neurobasal plus media to 1x final concentration to use as complete neurobasal media.
6. Discard the PO in the T-25 flasks and rinse the flasks three times with 5 mL of 1x PBS.
7. Carefully decant to remove the supernatant and resuspend the cell pellet by gently pipetting up and down 2-3 mL of complete neurobasal media. Determine the cell concentration and total cell numbers with a hemocytometer and trypan blue. Expect up to 1.0 x 10⁷ cells per embryo.
8. Plate the cells in the PO-coated T-25 flasks with approximately 2.0 x 10⁷ cells per flask in 5 ml of complete neurobasal media. Incubate the cell culture at 37 °C in a 5% CO₂ incubator.
9. Perform a half media change every 2-3 days by replacing 2.5 mL of media from the culture flask with freshly made complete neurobasal media.

3. Co-culture of primary neurons and microglia

NOTE: All subsequent steps are to be done in a sterile biosafety cabinet.

Seeding neurons in 96-well plates
1. Add 100 µL of 10 µg/mL PO per well and incubate the flasks for at least 1 h at 37 °C to coat the wells with PO. Wash the plate with 1x PBS 3 times before seeding cells and aspirate any remaining liquid after the last wash.
2. Neurons grown in T-25 flasks are harvested for co-culture after 2-5 days in culture. Replace the media with 5 mL of 1x Versene solution with 0.25% trypsin and 1 mg/mL DNase I to detach neurons from the flask. Place the flasks inside the incubator for 5-6 min for digestion and gently swirl the flask every 2-3 minutes to aid cell detachment.
3. Take the flask out of the incubator and check if cells are detached under a microscope. Add 0.5 mL of heat-inactivated FBS to neutralize the trypsin and DNase I. Transfer cells to a 15 mL conical tube. Wash the flask with 5 mL of complete neurobasal media once to maximize collected neurons. Collect all cells in the same 15 mL conical tube.
4. Centrifuge the cell suspension at 300 x g for 5 min at 4 °C. Discard the supernatant and gently resuspend the cell pellet in 2-3 mL of complete neurobasal media.
5. Count cells with a hemocytometer and add complete neurobasal media to achieve a final concentration of 7.5 x 10⁵ neurons/mL. Seed 100 µL of the cell suspension to each well in the PO-coated 96-well plate to obtain 7.5 x 10⁴ neurons/well. A yield of 5-6 million neurons per flask is expected.
6. Incubate the neurons in a 96-well plate overnight. Add 100 µL of complete neurobasal media to each well the next day.
  NOTE: Once neurons display appropriate neurite process growth (around day 3-4 after seeding in 96-well plates), they are ready for co-culture with microglia.
Isolation and microglia-neuronal co-culture
1. Between 8-10 days of mixed glia cultivation, collect microglia by gently washing the T-75 flasks with glia culture media in each flask with a 10 mL pipette, and transfer the cells and media into a sterile 50 mL conical tube. Wash the flasks again with 10 mL fresh glia culture media to detach additional microglia from the flasks and collect the media. A yield of 7.5 x 10⁵ microglia per flask is expected.
2. Add 20 mL of fresh glia culture media supplemented with 20 ng/mL MCSF to each flask. Microglia may be harvested once or twice more if the mixed glia culture is maintained.
3. Centrifuge the cell collection at 300 x g for 5 min at 4 °C. Carefully remove the supernatant and gently resuspend the pellet in 2 mL of complete neurobasal media.
4. Count the cells with a hemocytometer and trypan blue. Add complete neurobasal media to a final concentration of 2.5 x 10⁵ cells/mL.
5. From the 96-well neuronal culture plate, remove 100 µL of neurobasal media and add 100 µL of 2.5 x 10⁵ cells/mL cell suspension to seed 2.5 x 10⁴ microglia per well of 7.5 x 10⁴neurons. Incubate the culture overnight at 37 °C in a 5% CO₂ incubator to allow microglia to settle. The co-culture may now be used for downstream experiments.

Representative Results

A flowchart showing the key steps of the mixed glia culture for microglia is shown in Figure 1A. Overall, sparse cells and excessive cellular debris are expected on day 1 (Figure 1B). By day 4, increased cell number should be observed, especially with the generation of adherent astrocytes, as indicated by their elongated morphology (Figure 1C). A few microglia may be observed on top of the astrocytes or as small round cells floating in media. By day 8, a confluent layer of astrocytes will form the adherent layers of cells, whereas microglia will appear as round and bright cells that are semi-attached to the astrocytes or floating in media (Figure 1D). If microglial density is low by day 8, the culture may be incubated for additional days before harvest.

A flow chart showing the key steps of the neuronal culture is shown in Figure 2A. In a healthy culture, neurons will attach and form neurite processes after 1-3 days in vitro (Figure 2B). Similarly, upon seeding neurons into a 96-well plate, they will appear round initially, but by day 3, neurons should become adherent and form neurite processes that connect with neighboring neurons (Figure 2C). Conversely, unhealthy neuron cultures will contain cell clumps and cell debris, which can result from deficient cell dissociation or mishandling of neurons (Figure 2D).

A flow chart showing the key steps of the microglia-neuronal co-culture protocol is shown in Figure 3A. After adding 2.5 x 10⁴ microglia to 7.5 x 10⁴ neurons, round and large microglia should appear on top or between the neurons and neurite processes (Figure 3B). To further visualize the microglia-neuronal co-culture, microglia and neurons alone or in co-culture were labeled with ionized calcium-binding adaptor molecule 1 (IBA-1, red) and tubulin βIII (TUJ1, green) antibodies³⁷ for immunofluorescence microscopy (Figure 3C–F).

Figure 1: Overview of primary microglia culture. (A) Flow chart of the key steps in generating a mixed glia culture. (B–D) Representative 10x bright-field microscope images of mixed glia culture on day 1 (B), day 4 (C), and day 8 (D). Scale bars: 100 µm. Please click here to view a larger version of this figure.

Figure 2: Overview of primary neuron culture. (A) Flow chart of the key steps in generating neurons. (B) Representative 10x bright field microscope images showing neurons cultured in T-25 flasks. (C) Representative 10x bright field microscope of neurons cultured in a 96-well plate. (D) Representative 10x bright field microscope of an unhealthy neuronal cluster in a 96-well plate. Scale bars: 100 µm. Please click here to view a larger version of this figure.

Figure 3: Overview of microglia-neuron co-culture. (A) Flow chart of the key steps for the microglia-neuronal co-culture. (B) Representative 10x bright-field microscope images of microglia-neuronal co-culture in 96-well plates. Scale bars: 100 µm. (C,D) Representative immunofluorescence microscopy images of neurons alone (C), microglia alone (D), day 2 neuron and microglia co-culture (E), or 12-day neuron and microglia co-culture (F) in a 96-well plate. Microglia and neurons are labeled with IBA-1 (red) and tubulin βIII (green), respectively. Nuclei are stained with DAPI (blue). Immunofluorescence images were acquired using a 25x, 0.8 NA, water immersion objective with a widefield fluorescence microscope. Scale bars: 50 µm. Please click here to view a larger version of this figure.

Discussion

This article describes a protocol for isolating and culturing mouse primary neurons and primary microglia, which are subsequently used to establish a microglia-neuronal co-culture that can be used to study how microglia and neuron interactions regulate their cellular health and function. This relatively simple and accessible approach can provide critical insights into the mechanisms and functional outcomes of microglia neuron interactions in the CNS.

To achieve an optimal co-culture, several critical aspects in this protocol require extra care. During brain isolations (steps 1.2.6 and 2.2.7), meninges attached to the surface of both the embryonic and post-natal brains should be removed completely before collecting the tissues. Like other neuroglia protocols, incomplete removal of the meninges may result in cell culture becoming contaminated with endothelial cells or blood-derived cells⁴⁰^,⁴¹^,⁴². Additionally, when culturing primary neurons (steps 2.3.7 and 3.1.4), it is crucial to generate a single-cell suspension and avoid cell clumps before seeding neurons into flasks or 96-well plates. Neuronal aggregates will not properly adhere in culture and will lead to excessive neuronal stress and cell death. In addition, extended treatment of neurons with trypsin when attempting to dissociate them from T-25 flasks may cause unwanted cell death (step 3.1.2). To mitigate this, neurons may be directly seeded in 96-well plates at the desired cell density following dissociation from embryonic brains (step 2.3.7). Moreover, in this protocol, a 3:1 neuron: microglia ratio was selected to partially represent an elevated amount of microglia that may be present in the CNS microenvironment during neuroinflammation. Alternatively, the ratio of cells in the co-culture may be adjusted per experimental need. In addition, the timepoint selected for co-culture neurons may be extended if one aims to assess more mature neuronal states.

Notably, when dissociating neurons from T-25 flasks to 96-well plates, using trypsin alone may result in incomplete dissociation and the formation of cell clumps instead of a monolayer culture in 96-well plates (Figure 2D). To address this issue, neurons can be dissociated from the T-25 flask using 1x Versene solution containing 1 mg/mL DNase I. Other factors contributing to neuronal aggregation and reduced viability include incomplete trituration of the cell suspension, inadequate neutralization of trypsin with heat-inactivated FBS, excessive centrifugation, or prolonged cell pellet formation. Therefore, ensuring proper tissue dissociation and the generation of a single-cell suspension are critical for establishing a healthy microglia and neuron co-culture.

This protocol aims to recapitulate microglia-neuronal interactions in vitro to better understand the functional importance of their interactions in the CNS. While other types of co-culture systems have been developed, such as culturing individual cell populations on different surfaces to allow for independent treatment of two cell populations⁴³^,⁴⁴, this mixed culture system enables direct and close contact of microglia and neurons. In addition, when maintained properly through half media changes every 3 days, this mixed culture system can remain healthy from 12 days (Figure 3F) to potentially 3 weeks, allowing for long-term studies of neuronal-microglia interactions⁴⁵.

While the relative simplicity of the co-culture system is advantageous for accessibility, a key limitation of this approach is that the two-dimensional cell culture does not fully represent the CNS tissue environment. Indeed, a tri-culture of microglia, neurons, and astrocytes is shown to mimic in vivo neuroinflammatory response more reliably than microglia or astrocyte-neuronal co-culture⁴⁶^,⁴⁷. Furthermore, recent developments in brain organoid systems with microglia and other glial cells, including astrocytes and oligodendrocytes, are more suitable for studying three-dimensional (3D) tissue architecture and interactions of the CNS⁴⁸. Similarly, a human 3D triculture system comprising neurons, microglia, and astrocytes is shown to be effective in modeling Alzheimer's disease condition⁴⁹. Other human microglia-neuron co-cultures may be more physiologically relevant to neurodegenerative disease studies. For example, the co-culture of microglia and motor neurons derived from human induced pluripotent stem cells⁵⁰ may be useful for studying diseases such as amyotrophic lateral sclerosis⁵¹. Nevertheless, given the relative abundance and low cost of generating primary neurons and microglia from mice, assessing microglia-neuronal co-culture in 96-well plates can be a high throughput approach to studying the mechanisms and functional implications of microglia-neuronal interactions. Downstream assays may include using live cell imaging to determine cellular functional changes in real-time, RNA sequencing to determine cellular transcriptional changes, immunofluorescence microscopy to determine cellular phenotypic changes, mass spectrometry to determine cellular and proteomic changes, or enzyme-linked immunosorbent assay and western blot to measure protein secreted into the co-culture supernatant.

As microglia are important mediators of CNS health and diseases, this co-culture can also be used to investigate the effect of disease-related molecules and compounds on microglia-neuronal bi-directional communication. In the context of MS, activated microglia may take on a destructive role, contributing to the worsening of pathology⁵²^,⁵³. For example, they may secrete pro-inflammatory cytokines and reactive oxygen species, as well as promote astrocyte activation⁴⁵. This protocol may help to further interrogate the harmful role of microglia by treating microglia in the co-culture with inflammatory cytokines found elevated in MS and assessing the outcomes of neuron health. Alternatively, this microglia-neuronal co-culture has been used to show the protective effect of microglia on neurons in response to oxidized phosphatidylcholine, an oxidative product discovered in MS⁵⁴. Moreover, neuronal apoptosis remodels microglia gene expression during development by promoting microglia survival and phagocytosis⁵⁵. Therefore, this protocol may also help to determine the impact of neuronal death on microglia phenotype and function.

In summary, this protocol describes the generation and co-culturing of primary mouse-derived neurons and microglia in vitro. The microglia-neuronal co-culture set up in 96-well plates can be a high throughput approach to interrogate the mechanisms and function of microglia interactions with neurons in the context of CNS health and disease, including MS.

Declarações

The authors have nothing to disclose.

Acknowledgements

JP acknowledges funding support from the Natural Sciences and Engineering Research Council of Canada and the University of Saskatchewan College of Medicine. YD acknowledges funding support from the University of Saskatchewan College of Medicine Startup Fund, the Natural Sciences and Engineering Research Council of Canada Discovery Grant (RGPIN-2023-03659), MS Canada Catalyst Grant (1019973), Saskatchewan Health Research Foundation Establishment Grant (6368), and Brain Canada Foundation Future Leaders in Canadian Brain Research Grant. Figure 1A, Figure 2A, and Figure 3A were created with BioRender.com.

Materials

10 cm Petri dish	Fisher	07-202-011	Sterile
1x Versene	Gibco	15040-066
B-27 Plus Neuronal Culture System	Gibco	A3653401
Dissection microscope	VWR
DNase I	Roche	11284932001
Dulbecco’s Modified Eagle Medium (DMEM)	Gibco	11960-044
Fetal Bovine Serum	ThermoFisher Sci	12483-020
HBSS (10x)	Gibco	14065-056
Hemacytometer	Hausser Scientific	1475
HEPES	ThermoFisher Sci	15630080
Leibovitz’s L-15 Medium (1x)	Fisher Scientific	21083027
Macrophage colony stimulating factor	Peprotech	315-02
Micro-Forceps	RWD	F11020-11	Autoclaved/Sterile
Non-essential amino acids	Cytiva	SH3023801
PBS (10x)	ThermoFisher Sci	AM9625
Penicillin Streptomycin Glutamine (100x)	Gibco	103780-16
Poly-L-ornithine hydrobromide	Sigma	P3655-100MG
Sodium pyruvate (100 mM)	Gibco	11360-070
Spring scissors	RWD	S11008-42	Autoclaved/Sterile
Surgical blade	Feather	08-916-5D	Sterile
T-25 flasks	Fisher	10-126-9
T-75 flasks	Fisher	13-680-65
Tissue forceps	Codman	30-4218	Autoclaved/Sterile
Tissue scissors	RWD	S12052-10	Autoclaved/Sterile
Trypan Blue	Thermofisher Sci	15250-061
Trypsin (2.5%)	ThermoFisher Sci	15090046
Widefield Immunofluorescence Microscope	Zeiss

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