Related Experiment Video
Updated: Dec 11, 2025
Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination
Published on: April 5, 2022
Methodological comparison of FACS and MACS isolation of enriched microglia and astrocytes from mouse brain
1Shenzhen Key Laboratory for Neuronal Structural Biology, Biomedical Research Institute, Shenzhen Peking University - The Hong Kong University of Science and Technology Medical Center, Guangdong Province, Shenzhen, China.
Abstract:
Microglia and astrocytes, the two innate cells in CNS, are thought to protect and remodel of synapses for proper maintenance and plasticity of neuronal circuits. The two types of cells are the major responders by producing and releasing inflammatory mediators. Isolation of microglia and astrocytes from CNS tissue provides a powerful tool to study basic cell biology and examine the effects of in vivo treatments on microglia and astrocytes immunophenotype and function. The widely used approach of enrichment microglia and astrocytes from CNS was MACS (Magnetic activated cell sorting) and FACS (Fluorescence activated cell sorting). Here we described an optimized protocol of enzymatic dissociation generating single cell suspensions from brain tissue. Then the ability of the two methods to isolate microglia and astrocytes from brain dissociated cells was compared. Both MACS and FACS processing could obtain microglia and astrocytes with high viability (>85%). Microglia sorted by MACS comprises a slight myeloid cells contamination but with a little bit higher efficiency than that sorted by FACS. MACS processing was faster than FACS for either single or multiple samples. ACSA2 can be used to isolate astrocytes from both postnatal and adult brain, and is more suitable for purify astrocytes from newborn. FACS could get purer microglia which is helpful for deep sequencing and other related research. ACSA2 is a good marker of astrocytes.
Insights
Magnetic activated cell sorting (MACS) and Fluorescence activated cell sorting (FACS) effectively isolate microglia and astrocytes from the central nervous system (CNS). MACS offers faster processing and higher efficiency, while FACS provides purer microglia for advanced research.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia and astrocytes are key innate immune cells in the central nervous system (CNS).
- These glial cells play vital roles in synaptic maintenance, neuronal plasticity, and immune responses within the brain.
- Studying these cells requires efficient isolation methods to analyze their function and response to treatments.
Purpose of the Study:
- To compare the efficacy of Magnetic Activated Cell Sorting (MACS) and Fluorescence Activated Cell Sorting (FACS) for isolating microglia and astrocytes from brain tissue.
- To optimize a protocol for enzymatic dissociation of CNS tissue into single-cell suspensions for subsequent cell sorting.
- To evaluate the purity, viability, and efficiency of both MACS and FACS methods for obtaining these specific glial cell populations.
Main Methods:
- Enzymatic dissociation of brain tissue to generate single-cell suspensions.
- Isolation of microglia and astrocytes using Magnetic Activated Cell Sorting (MACS).
- Isolation of microglia and astrocytes using Fluorescence Activated Cell Sorting (FACS), utilizing ACSA2 as an astrocyte marker.
Main Results:
- Both MACS and FACS methods achieved high cell viability (>85%) for isolated microglia and astrocytes.
- MACS processing was faster and yielded slightly higher isolation efficiency for microglia, albeit with minor myeloid cell contamination.
- FACS provided purer microglia populations, beneficial for downstream applications like deep sequencing, and demonstrated ACSA2 as a reliable astrocyte marker across different ages.
Conclusions:
- Both MACS and FACS are viable methods for isolating microglia and astrocytes from the CNS, each with distinct advantages.
- MACS is a rapid and efficient choice for general isolation, while FACS offers superior purity for specialized research requiring highly pure cell populations.
- The optimized enzymatic dissociation protocol combined with MACS or FACS enables robust study of glial cell biology and function in the CNS.
