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Proposed practical protocol for flow cytometry analysis of microglia from the healthy adult mouse brain: Systematic
Sanja Srakočić1, Paula Josić1, Sebastijan Trifunović1
1Croatian Institute for Brain Research, School of Medicine, University of Zagreb, Zagreb, Croatia.
Abstract:
The aim of our study was to systematically analyze the literature for published flow cytometry protocols for microglia isolation and compare their effectiveness in terms of microglial yield, including our own protocol using sucrose for myelin removal and accutase for enzymatic digestion. For systematic review, the PubMed was searched for the terms "flow cytometry," "microglia," "brain," and "mice." Three different myelin removal methods (Percoll, sucrose, and no removal) and five protocols for enzymatic digestion (accutase, dispase II, papain, trypsin, and no enzymatic digestion) were tested for the effectiveness of microglia (CD11b+CD45int cell population) isolation from the adult mouse brain using flow cytometry. Qualitative analysis of the 32 selected studies identified three most commonly used myelin removal protocols: Percoll, the use of myelin removal kit, and no removal. Nine enzymatic digestion protocols were identified, from which we selected dispase II, papain, trypsin, and no enzymatic digestion. A comparison of these myelin removal methods and digestion protocols showed the Percoll method to be preferable in removal of non-immune cells, and superior to the use of sucrose which was less effective in removal of non-immune cells, but resulted in a comparable microglial yield to Percoll myelin removal. Digestion with accutase resulted in one of the highest microglial yields, all while having the lowest variance among tested protocols. The proposed protocol for microglia isolation uses Percoll for myelin removal and accutase for enzymatic digestion. All tested protocols had different features, and the choice between them can depend on the individual focus of the research.
Insights
This study systematically reviewed flow cytometry protocols for isolating microglia (CD11b+CD45int) from mouse brains. Percoll for myelin removal and Accutase for digestion yielded high microglial populations with low variance.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia, the immune cells of the central nervous system, play crucial roles in brain health and disease.
- Accurate isolation of microglia is essential for studying their function and response to stimuli.
- Existing protocols for microglia isolation using flow cytometry vary in effectiveness.
Purpose of the Study:
- To systematically analyze and compare published flow cytometry protocols for microglia isolation from adult mouse brains.
- To evaluate the effectiveness of different myelin removal and enzymatic digestion methods on microglial yield and purity.
- To identify an optimal protocol for high-yield, low-variance microglia isolation.
Main Methods:
- Systematic literature review of PubMed using keywords: "flow cytometry," "microglia," "brain," and "mice."
- Comparative analysis of three myelin removal methods (Percoll, sucrose, no removal) and five enzymatic digestion protocols (accutase, dispase II, papain, trypsin, no digestion).
- Evaluation of microglial yield (CD11b+CD45int population) and purity based on selected studies and experimental testing.
Main Results:
- Percoll demonstrated superior non-immune cell removal compared to sucrose, though both yielded comparable microglial numbers.
- Accutase enzymatic digestion resulted in one of the highest microglial yields with the lowest protocol variance.
- The study proposes a protocol combining Percoll for myelin removal and Accutase for enzymatic digestion.
Conclusions:
- Protocol choice for microglia isolation depends on specific research needs and priorities.
- Combining Percoll and Accutase offers an effective method for high-yield microglia isolation with flow cytometry.
- Systematic comparison aids researchers in selecting optimal protocols for microglia analysis.
