Primary Microglia Culture and Isolation Service

Microglia are the resident immune cells of the central nervous system (CNS) and play a crucial role in maintaining homeostasis, responding to injury, and mediating neuroinflammation. They are considered the first line of defense in the CNS and have a significant impact on various neurological diseases.

As is well documented, dysregulation of microglial function has been well established in pathologies associated with neurodegeneration, including Alzheimer's disease, Parkinson's disease, multiple sclerosis, and Huntington's disease. Primary microglia cultures allow for targeted investigation of the impact of various compounds on microglial, typically harvested from embryonic or early postnatal mouse pups. In addition to mouse microglia, Creative Biolabs offers primary neuronal cultures derived from rat and human as well. We have an in-depth explanation of primary microglia culture methods and our services here.

Isolation and Purification of Primary Microglia

Our Neuros team excels at preparing primary microglial monocultures from the brains of human and rodents. The cell culture protocols can be flexibly performed depending on species and ages of animals. Below is the workflow for establishing primary microglial cultures from the brains of neonatal mice.

We can also isolate and purify primary microglia from adult animals using flow cytometry.

Applications

• Neuroinflammation

Primary microglia are essential for studying the mechanisms of neuroinflammation. Researchers can investigate how microglia respond to different stimuli, such as pathogens, toxins, or injury, and the signaling pathways involved in the inflammatory response.

• Neurodegenerative Disease Modeling

Microglia have been implicated in several neurodegenerative diseases, including Alzheimer's, Parkinson's, and multiple sclerosis. Primary microglia can be used to model and investigate disease mechanisms, such as amyloid-beta plaque formation or the role of microglial activation in neurodegeneration.

• Cellular Interactions

Microglia interact with various cell types in the CNS, including neurons, astrocytes, and oligodendrocytes. Using primary microglia in co-culture systems allows researchers to explore these interactions and their implications for CNS health and disease.

• Phagocytosis and Cellular Clearance

Microglia are responsible for clearing cellular debris and dead cells from the CNS. Research using primary microglia can help elucidate their mechanisms of phagocytosis and the effects of impaired clearance on neurological health.

• Neurodevelopmental Research

Studies of microglial roles during brain development can reveal how these cells influence neuronal maturation, synapse formation, and pruning, which are critical processes in cognitive function.

• Drug Development and Toxicology

Primary microglia can be utilized to assess the effects of novel therapeutics or toxic substances on CNS immune responses. This includes studying the safety and efficacy of drugs aimed at modulating microglial activity.

Downstream Testing

Primary microglia functional assays are crucial for understanding the role of microglia in the central nervous system (CNS), including their involvement in neuroinflammation, synaptic pruning, and response to injury or disease. Here are some common assays used to evaluate the functional properties of primary microglia:

• Proinflammatory Cytokines and Chemokines Assessment
  
  • Measure the secretion levels of key proinflammatory cytokines (e.g., TNF-α, IL-1β, IL-6) and chemokines using enzyme-linked immunosorbent assay (ELISA) to evaluate the pro-inflammatory response of microglia after LPS treatment.
  • Utilize quantitative PCR (qPCR) to assess mRNA expression of various cytokines and chemokines, providing insights into the transcriptional regulation during inflammatory responses.
  • Employ Western blotting to detect and quantify the protein levels of specific signaling molecules involved in microglial activation and inflammation.
• Phagocytosis Assay
  
  • Assess phagocytosis by incubating primary microglia with fluorescent latex beads or fluorescent Aβ42 and measuring uptake using flow cytometry or confocal imaging.
  • Use live-cell imaging techniques to visualize and record the dynamics of phagocytosis in real-time, providing insights into the kinetics and efficiency of microbial clearance.
  • Investigate the involvement of specific receptors (e.g., CD36, scavenger receptors) in the phagocytic process through pharmacological inhibition or gene knockout studies.
• Co-culture Systems
  
  • Establish co-culture systems with neurons or astrocytes to evaluate the interactions between different cell types and their impact on microglial activation and function.
  • Assess cytokine release profiles in the co-culture supernatants to understand the paracrine signaling mechanisms influencing microglial behavior.
  • Use immunofluorescence staining to visualize the morphological changes in microglia that occur in response to neighboring cell types, indicating their activation state.
• Oxidative Stress Assay
  
  • Malondialdehyde (MDA) and Superoxide Dismutase (SOD) assay kits can be used to detect intracellular oxidative stress levels. Measure the level of MDA and SOD in primary microglia cultures to assess the degree of oxidative stress. Essentially, higher MDA level and lower SOD activity indicate increased oxidative damage in primary microglia cultures.
  • After various treatments, primary microglia cultures can be incubated with the fluorescent probe DCFH-DA, dihydroethidium, or MitoSOX Red mitochondrial superoxide indicator and then analyzed by flow cytometry to determine the levels of intracellular ROS, O_2⁻, and mitochondrial O_2⁻.
• Cell Migration Assay
  
  • The assessment of microglial migration can be effectively conducted using specific assays. The transwell migration assay, which utilizes chemoattractants, is particularly suitable for evaluating the migratory behavior of primary microglial monocultures.

Reference

1. Layman, Elsie et al. "Protocol for assessing phagocytosis activity in cultured primary murine microglia." STAR Protoc. 2022;3(4):101881. Distributed under Open Access license CC BY 4.0, without modification.