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Updated: Mar 26, 2026
Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates
Published on: January 30, 2014
Characterization of a novel adult murine immortalized microglial cell line and its activation by amyloid-beta
Ryan C McCarthy1, Dah-Yuu Lu2,3, Ahmed Alkhateeb4,5
1Department of Genetics and Complex Diseases, Harvard School of Public Health, 655 Huntington Avenue, Boston, MA, 02115, USA. rmccarth@hsph.harvard.edu.
Background:
Alzheimer's disease is associated with amyloid-beta (Aβ)-induced microglia activation. This pro-inflammatory response promotes neuronal damage, and therapies are sought to limit microglial activation. Screening efforts to develop new pharmacological inhibitors require a robust in vitro cell system. Current models lack significant responses to Aβ, and their use in examining age-related neurodegenerative diseases is questionable. For example, the commonly used BV-2 microglial line was derived from embryonic mononuclear cells and its activation by various stimuli is limited. To this end, we have established a new immortalized microglial (IMG) cell line from adult murine brain. The objective of this study was to characterize Aβ-induced activation of IMG cells, and here, we demonstrate the ability of cannabinoids to significantly reduce this inflammatory response.
Methods:
Microglial cells derived from adult murine brain were immortalized via infection with the v-raf/v-myc retrovirus under conditions that selectively promote microglia growth. The presence or absence of markers CD11b and F4/80 (microglial), NeuN (neuronal), and GFAP (astrocytic) was assessed by immunofluorescence microscopy and western blotting. Using IMG and BV-2 cells, levels of pro- and anti-inflammatory transcripts in response to extracellular stimuli were determined by quantitative PCR (qPCR). Phagocytosis of fluorescent beads and fluorescein isothiocyanate (FITC)-labeled Aβ oligomers was assessed using flow cytometry and fluorescence microscopy. FITC-Aβ uptake was quantified using a fluorescence plate reader. The ability of cannabinoids to mitigate Aβ-induced expression of inducible nitric oxide synthase (iNOS) was evaluated.
Results:
IMG cells express the microglial markers CD11b and F4/80 but not NeuN or GFAP. Relative to BV-2 cells, IMG cells increased iNOS (>200-fold) and Arg-1 (>100-fold) in response to pro- and anti-inflammatory stimuli. IMG cells phagocytose foreign particles and Aβ oligomers, with the latter trafficked to phagolysosomes. Aβ-induced activation of IMG cells was suppressed by delta-9-tetrahydrocannabinol and the CB2-selective agonist JWH-015 in a time- and concentration-dependent manner.
Conclusions:
IMG cells recapitulate key features of microglial cell activation. As an example of their potential pharmacological use, cannabinoids were shown to reduce activation of Aβ-induced iNOS gene expression. IMG cells hold promising potential for drug screening, mechanistic studies, and functional investigations directed towards understanding how Aβ interacts with microglia.
Insights
A new immortalized microglial cell line (IMG) effectively models Alzheimer's disease-related inflammation. Cannabinoids significantly reduce amyloid-beta-induced inflammatory responses in these cells, offering a promising tool for drug discovery.
Area of Science:
- Neuroscience
- Immunology
- Pharmacology
Background:
- Alzheimer's disease (AD) involves amyloid-beta (Aβ)-induced microglial activation, a process contributing to neuroinflammation and neuronal damage.
- Current in vitro models, like the BV-2 microglial line, exhibit limited responses to Aβ, hindering effective drug screening for AD therapies.
- A novel immortalized microglial (IMG) cell line derived from adult murine brain was developed to address limitations in existing models.
Purpose of the Study:
- To characterize the Aβ-induced activation of the newly established IMG cell line.
- To evaluate the potential of IMG cells as a model for studying neuroinflammation in AD.
- To investigate the efficacy of cannabinoids in mitigating Aβ-induced inflammatory responses in IMG cells.
Main Methods:
- Immortalization of microglial cells from adult murine brain using v-raf/v-myc retrovirus.
- Characterization of IMG cells using immunofluorescence and western blotting for microglial, neuronal, and astrocytic markers.
- Quantitative PCR (qPCR) to assess inflammatory and anti-inflammatory transcript levels.
- Flow cytometry and fluorescence microscopy to evaluate phagocytosis of beads and Aβ oligomers.
- Assessment of cannabinoid effects on Aβ-induced inducible nitric oxide synthase (iNOS) expression.
Main Results:
- IMG cells express microglial markers (CD11b, F4/80) and not neuronal (NeuN) or astrocytic (GFAP) markers.
- IMG cells demonstrated significantly higher induction of iNOS (>200-fold) and Arg-1 (>100-fold) compared to BV-2 cells in response to stimuli.
- IMG cells effectively phagocytosed Aβ oligomers, trafficking them to phagolysosomes.
- Delta-9-tetrahydrocannabinol and JWH-015 (a CB2 agonist) significantly suppressed Aβ-induced iNOS expression in IMG cells in a dose- and time-dependent manner.
Conclusions:
- The IMG cell line accurately recapitulates key features of microglial activation relevant to AD.
- Cannabinoids demonstrate potential in reducing Aβ-induced inflammatory gene expression, highlighting their therapeutic relevance.
- IMG cells represent a valuable tool for drug screening, mechanistic studies, and functional investigations into Aβ-microglia interactions in neurodegenerative diseases.
