Dissociation of neonatal and adult mice brain for simultaneous analysis of microglia, astrocytes and infiltrating lymphocytes by flow cytometry

Calvo, Belen; Rubio, F. Bazzino; Fernández, Miriam; Tranque, Pedro

doi:10.1016/j.ibror.2019.12.004

Cited by 26 publications

(20 citation statements)

References 51 publications

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“…Given the extent of CD45 and CD11b expression, the resident microglia (CD11b hi /CD45 lo /CCR2 − ) and infiltrating macrophages (CD11b lo /CD45 hi /CCR2 + ) in the injured spinal cord were distinguished by FACS analysis (Fig. 3 H–L) in accordance with previous study [ 46 ]. We found that there was no difference in the amount of microglia and recruited macrophages among all groups at day 14 after SCI in the injury site, as demonstrated by dot plots and quantitative analyses (Additional file 1 : Fig.…”

Section: Resultssupporting

confidence: 87%

Interleukin-10 genetically modified clinical-grade mesenchymal stromal cells markedly reinforced functional recovery after spinal cord injury via directing alternative activation of macrophages

et al. 2022

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Background After spinal cord injury (SCI), dysregulated or nonresolving inflammatory processes can severely disturb neuronal homeostasis and drive neurodegeneration. Although mesenchymal stromal cell (MSC)-based therapies have showed certain therapeutic efficacy, no MSC therapy has reached its full clinical goal. In this study, we examine interleukin-10 (IL10) genetically modified clinical-grade MSCs (IL10-MSCs) and evaluate their clinical safety, effectiveness, and therapeutic mechanism in a completely transected SCI mouse model. Methods We established stable IL10-overexpressing human umbilical-cord-derived MSCs through electric transduction and screened out clinical-grade IL10-MSCs according to the criteria of cell-based therapeutic products, which were applied to mice with completely transected SCI by repeated tail intravenous injections. Then we comprehensively investigated the motor function, histological structure, and nerve regeneration in SCI mice, and further explored the potential therapeutic mechanism after IL10-MSC treatment. Results IL10-MSC treatment markedly reinforced locomotor improvement, accompanied with decreased lesion volume, regeneration of axons, and preservation of neurons, compared with naïve unmodified MSCs. Further, IL10-MSC transplantation increased the ratio of microglia to infiltrated alternatively activated macrophages (M2), and reduced the ratio of classically activated macrophages (M1) at the injured spinal cord, meanwhile increasing the percentage of Treg and Th2 cells, and reducing the percentage of Th1 cells in the peripheral circulatory system. In addition, IL10-MSC administration could prevent apoptosis and promote neuron differentiation of neural stem cells (NSCs) under inflammatory conditions in vitro. Conclusions IL10-MSCs exhibited a reliable safety profile and demonstrated promising therapeutic efficacy in SCI compared with naïve MSCs, providing solid support for future clinical application of genetically engineered MSCs.

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Section: Resultssupporting

confidence: 87%

Interleukin-10 genetically modified clinical-grade mesenchymal stromal cells markedly reinforced functional recovery after spinal cord injury via directing alternative activation of macrophages

et al. 2022

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“…The Percoll gradient is an important step in the microglial isolation process, as this technique can effectively remove myelin and other debris and separate cells based on size. 19 To help identify the microglial layer following centrifugation, we have provided a picture of the tube at this step (Figure 4). The microglial layer is a subtle, gray-white layer located at the interface of the lighter pink 30% Percoll layer and the darker pink 70% Percoll layer.…”

Section: Resultsmentioning

confidence: 99%

“…The homogenate was filtered with a 70 µm cell strainer, and the cell suspension was subjected to Percoll gradient centrifugation. The methods for the 70%/37%/30% gradient 18 and the 70/30% gradient 19 have been previously published.…”

Section: Methodsmentioning

confidence: 99%

Development of a novel purification protocol to isolate and identify brain microglia

Doughty

Rajpurohit

Trang

et al. 2022

Exp Biol Med (Maywood)

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Microglia, the tissue-resident macrophage of the central nervous system (CNS), play a paramount role in brain health and disease status. Here, we describe a novel method for enriching and isolating primary microglia from mouse brain tissue. This isolation method yields a high number of cells from either young or adult mice, and importantly, maintains the health and function of the cells for subsequent cell culture. We also describe flow cytometry methods using novel cell surface markers, including CX3CR1 and Siglec-H, to specifically label microglia while avoiding other bone marrow and/or non-CNS derived macrophages and monocytes, which has been historically difficult to achieve. As microglia are crucial in multiple aspects of biology, such as in normal brain development/function, immune response, neurodegeneration, and cancer, this isolation technique could greatly benefit a wide range of studies in human CNS biology, health, and disease mechanisms. Being able to isolate a largely pure population of microglia could also allow for a more comprehensive understanding of their functional dynamics and role in disease mechanisms, advancement of potential biomarkers, and development of novel therapeutic targets to improve prognosis and quality of life in multiple diseases.

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“…The first protocol of myelin removal was performed in 15 mL centrifuge tubes with three different solution gradients of Percoll (Sigma Aldrich, St. Louis, Missouri, MO, USA) dissolved in 10X HBSS: 30% (4 mL), 37% (4 mL) and 70% (4 mL) ( Calvo et al, 2020 ). After debris removal, the cells were dissolved in 37% isotonic Percoll solution.…”

Section: Methodsmentioning

confidence: 99%

“…Flow cytometry is an indispensable method for the quantification and identification of the cells in any tissue sample, including brain samples containing microglia ( Bohlen et al, 2019 ; Brioschi et al, 2020 ; Calvo et al, 2020 ). When being prepared for flow cytometric analysis, already mechanically processed tissue is frequently digested with enzymes as chemical agents.…”

Section: Introductionmentioning

confidence: 99%

Proposed practical protocol for flow cytometry analysis of microglia from the healthy adult mouse brain: Systematic review and isolation methods’ evaluation

Srakočić

Josić

Trifunović

et al. 2022

Front. Cell. Neurosci.

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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.

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Dissociation of neonatal and adult mice brain for simultaneous analysis of microglia, astrocytes and infiltrating lymphocytes by flow cytometry

Cited by 26 publications

References 51 publications

Interleukin-10 genetically modified clinical-grade mesenchymal stromal cells markedly reinforced functional recovery after spinal cord injury via directing alternative activation of macrophages

Interleukin-10 genetically modified clinical-grade mesenchymal stromal cells markedly reinforced functional recovery after spinal cord injury via directing alternative activation of macrophages

Development of a novel purification protocol to isolate and identify brain microglia

Proposed practical protocol for flow cytometry analysis of microglia from the healthy adult mouse brain: Systematic review and isolation methods’ evaluation

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