Here, we present a protocol to isolate and purify primary microglia in animal models of demyelinating diseases, utilizing columnar magnetic-activated cell sorting.
Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). Microglia can be divided into resting state and activated state and can rapidly change state in response to the microenvironment of the brain. Microglia will be activated under different pathological conditions and exhibit different phenotypes. In addition, there are many different subgroups of activated microglia and great heterogeneity between different subgroups. The heterogeneity mainly depends on the molecular specificity of microglia. Studies have revealed that microglia will be activated and play an important role in the pathological process of inflammatory demyelination. To better understand the characteristics of microglia in inflammatory demyelinating diseases such as multiple sclerosis and neuromyelitis optica spectrum disorder, we propose a perilesional primary microglial sorting protocol. This protocol utilizes columnar magnetic-activated cell sorting (MACS) to obtain highly purified primary microglia and preserve the molecular characteristics of microglia to investigate the potential effects of microglia in inflammatory demyelinating diseases.
Microglia originate from yolk-sac progenitors, which reach the embryonic brain very early and participate in the development of the CNS1,2. For instance, they are involved in synaptic pruning3 and regulating axonal growth4. They secrete factors that promote neuronal survival and help neuronal localization5. At the same time, they are involved in removing abnormal cells and apoptotic cells to ensure normal brain development6. In addition, as the immune-competent cells of the brain, microglia continually surveil the brain parenchyma to clear dead cells, dysfunctional synapses, and cellular debris7. It has been demonstrated that microglial activation plays an important role in a variety of diseases, including inflammatory demyelinating diseases, neurodegenerative diseases, and brain tumors. Activated microglia in multiple sclerosis (MS) contribute to the differentiation of oligodendrocyte precursor cells (OPCs) and regeneration of myelin by engulfing myelin debris8.
In Alzheimer's disease (AD), accumulation of amyloid beta (Aβ) activates microglia, which affects the phagocytic and inflammatory functions of microglia9. Activated microglia in the glioma tissue, called glioma-associated microglia (GAM), can regulate the progression of glioma and ultimately affect the prognosis of patients10. The activation profoundly alters the microglial transcriptome, resulting in morphological changes, expression of immune receptors, increased phagocytic activity, and enhanced cytokine secretion11. There are different subsets of activated microglia in neurodegenerative diseases such as disease-associated microglia (DAM), activated response microglia (ARM), and microglial neurodegenerative phenotype (MGnD)8.
Similarly, multiple dynamic functional subsets of microglia also coexist in the brain in inflammatory demyelinating diseases12. Understanding the heterogeneity between different subsets of microglia is essential to investigate the pathogenesis of inflammatory demyelinating diseases and to find their potential therapeutic strategies. The heterogeneity of microglia mainly depends on the molecular specificity8. It is essential to describe the molecular alterations of microglia accurately for the study of the heterogeneity. Advances in single-cell RNA-sequencing (RNA-seq) technology have enabled the identification of the molecular characteristics of activated microglia in pathological conditions13. Therefore, the ability to isolate cell populations is critical for further investigation of these target cells under specific conditions.
Studies performed to understand the characteristics and functions of microglia are usually in vitro studies, since it has been found that large numbers of primary microglia can be prepared and cultured from mouse pup brains (1-3 days old), which attach to the culture flasks and grow on the plastic surface with other mixed glial cells. Subsequently, pure microglia can be isolated based on the different adhesiveness of mixed glial cells14,15. However, this method can only isolate microglia from the perinatal brain and takes several weeks. Potential variables in cell culture may influence microglial characteristics such as molecular expression16. Moreover, microglia isolated by these methods can only participate in in vitro experiments by simulating the conditions of CNS diseases and cannot represent the characteristics and functions of microglia in in vivo disease states. Therefore, it is necessary to develop methods for isolating microglia from the adult mouse brain.
Fluorescence-activated cell sorting (FACS) and magnetic separation are two widely used methods, although they have their own different limitations16,17,18,19. Their respective advantages and disadvantages will be contrasted in the discussion section. The maturation of MACS technology offers the possibility to rapidly purify cells. Huang et al. have developed a convenient method to label demyelinating lesions in brains20. Combining these two technical approaches, we propose a rapid and efficient columnar CD11b magnetic separation protocol, providing a step-by-step description to isolate microglia around demyelinating lesions in adult mouse brains and preserve the molecular characteristics of microglia. The focal demyelinating lesions were caused by the stereotactic injection of 2 µL of lysolecithin solution (1% LPC in 0.9% NaCl) in the corpus callosum 3 days before starting the protocol21. This protocol lays the foundation to perform the next step in in vitro experiments. Moreover, this protocol saves time and remains feasible for widespread use in various experiments.
All the animal procedures have been approved by the Institute of Animal Care Committee of Tongji Medical College, Huazhong University of Science and Technology, China.
1. Materials
2. Mice perfusion and dissection
3. Tissue dissociation
4. Debris removal
5. Magnetic separation of microglial cells
Microglia isolated using CD11b beads have high purity
Microglial cells around the lesions in demyelination mouse models were isolated using the above-mentioned protocol and tested by flow cytometry. Cells are fluorescently labeled with CD11b-fluorescein isothiocyanate (FITC) and CD45-allophycocyanin (APC) to determine microglia in flow cytometry according to the manufacturer's instructions. There are multiple literatures demonstrating that CD11b and CD45 antibodies are enough to check for the purity of isolated microglia19,22. The fluorescence compensation was completed on the flow cytometer using unstained and single-stained control samples. Cells were gated by forward scatter-height (FSC-H) and side scatter-height (SSC-H); single cells were gated by forward scatter-area (FSC-A) and FSC-H. The proportions of microglia (identified as CD11b+CD45Intermediate) before and after the magnetic separation were tested by flow cytometry. The back-loop technique was used to adjust the gating strategy of flow cytometry analysis by determining the gate for the microglia. Compared with approximately 3 × 104 cells and 6 × 103 microglia before magnetic separation of each mouse brain (Figure 3A), MACS generally yields approximately 2.5 × 103 cells and 2 × 103 microglia per mouse brain (Figure 3B), which was approximately 85% of all single cells. However, nearly 3% of myeloid cells (identified as CD11b+CD45high) were present in the MACS-separated cells and were difficult to remove from the CD11b population in this protocol. The purity of the isolated microglia can be increased to 95% using two magnetic columns (Supplemental Figure S1). MACS sorting could capture approximately 33% of microglia in the brain mixture sample.
Microglia isolated using CD11b beads have high cell viability
Fluorochrome 7-aminoactinomycin D (7-AAD) is often used to show the cell viability in the flow cytometry. 7-AAD+ cells represent dead cells19. The viability of MACS-separated microglia was approximately 95% by cell staining with 7-AAD (Figure 4).
Morphological analysis of microglia around demyelinating lesions sorted by MACS
Morphological analysis showed that microglia were not activated by MACS. Microglia exhibit a typical longitudinal bipolar cell body after MACS, which is similar to microglial morphology after FACS19. Microglia around demyelinating lesions will be activated in the model of LPC-induced demyelination21. Microglia stained by ionized calcium binding adaptor molecule 1 (Iba-1) showed typical amebic morphology of activated microglia in this protocol. P2ry12 is a typical molecule of microglial resting state. There is a small fraction of branched and P2ry12+ microglia before MACS due to the expansion of the extent of the dissociated lesions. However, the existence of P2ry12+ microglia after MACS also indicates that MACS will not activate microglia (Supplemental Figure S2).
Figure 1: Images of the focal demyelinating lesions labeled by the neutral red dye. Please click here to view a larger version of this figure.
Figure 2: Images of the three layers formed by centrifugation. The top two layers (layer 1 + layer 2) need to be removed in the debris removal process (see protocol step 4.5). Please click here to view a larger version of this figure.
Figure 3: Flow cytometry analysis of microglia isolated from demyelination lesions of adult mice before and after MACS. (A) Schematic representation of gating strategy used in flow cytometry analysis before MACS: FSC-H and SSC-H for selecting cells, FSC-A and FSC-H for selecting single cells, and CD11b-FITC and CD45-APC for selecting myeloid cells (up) and microglia (down). (B) Schematic representation of the gating strategy used in flow cytometry analysis sample after MACS. The proportion of microglia has increased significantly after MACS: FSC-H and SSC-H for selecting cells, FSC-A and FSC-H for selecting single cells, and CD11b-FITC and CD45-APC for selecting myeloid cells (up) and microglia (down). Abbreviations: SSC-H = side scatter-height; FSC-H = forward scatter-height; FSC-A = forward scatter-area; CD45-APC = allophycocyanin-labeled CD45; CD11b-FITC = fluorescein isothiocyanate-labeled CD11b; MACS = magnetic-activated cell sorting. Please click here to view a larger version of this figure.
Figure 4: FACS gating strategy for living/dead single cells after MACS. 7-AAD and SSC-H for selecting living cells after MACS. Abbreviations: SSC-H = side scatter-height; 7-AAD = 7-aminoactinomycin D; MACS = magnetic-activated cell sorting; FACS = fluorescence-activated cell sorting. Please click here to view a larger version of this figure.
Supplemental Figure S1: Flow cytometry analysis of microglia isolated from adult demyelination mice. (A) Schematic representation of the gating strategy used in flow cytometry analysis sample before MACS. (B) Schematic representation of the gating strategy used in flow cytometry analysis sample after MACS using two magnetic columns. (C) Single-parameter histogram of CD11b-FITC and CD45-APC in flow cytometry after MACS using two magnetic columns. Abbreviations: SSC-H = side scatter-height; FSC-H = forward scatter-height; FSC-A = forward scatter-area; CD45-APC = allophycocyanin-labeled CD45; CD11b-FITC = fluorescein isothiocyanate-labeled CD11b; MACS = magnetic-activated cell sorting; CD45Int = intermediate CD45 expression. Please click here to download this File.
Supplemental Figure S2: Immunofluorescence staining of microglia with Iba-1 and P2ry12 before and after MACS. (A) Images of microglia before MACS. (B) Images of isolated microglia after MACS. Scale bars = 20 µm. Abbreviations: MACS = magnetic-activated cell sorting; Iba-1 = ionized calcium binding adaptor molecule 1; DAPI = 4',6-diamidino-2-phenylindole. Please click here to download this File.
The protocol proposes a method to isolate microglia around the demyelinating lesions, which can help study the functional characteristics of microglia in inflammatory demyelinating diseases. Microglia captured using CD11b beads exhibit high purity and viability. Critical steps in the protocol include the precise localization of foci and optimal microglial purification. In protocol step 2.1, it is necessary to inject the NR solution 2 h before sacrificing the mouse to ensure that the lesions can be accurately displayed20. In protocol step 2.8, care was taken to place brain tissue sections on an ice box for dissection and complete this step as soon as possible, thereby improving cell viability. In step 2.9, it is necessary to select appropriate tissue as one sample for both enzyme digestion and debris removal. Step 3.7 cannot be skipped when the effect of debris removal is not satisfactory. In step 4.3, the addition of PBS must be gentle to form different layers, ensuring that as much debris is removed as possible. All the steps in the protocol, except enzymatic digestion, should be performed on ice to ensure cell viability and reduce the transcriptional response of the microglia. Each step in the protocol is mild and retains high viability, so it is unnecessary to use the dead cells removal solution22,23.
The enzymatic dissociation method for mouse brain in the protocol has proven suitable for single-cell RNA-seq23. Although the enzymes in the protocol are mild, Marsh et al. have found that various enzymatic hydrolysis protocols at 37 °C may significantly alter the transcriptome of microglia24. Setting a healthy control in the experiment for comparison can help to identify the differentially expressed genes of demyelination, thus eliminating the abnormal transcriptional response in the protocol. Meanwhile, toprevent this abnormal transcriptional state after enzymatic digestion, the protocol can be optimized by adding various transcription and translation inhibitors such as actinomycin D, triptolide and anisomycin at different steps. The use of these inhibitors can reveal the real gene expression of microglia in vivo without the setting of a control. Therefore, the addition of these inhibitors to the protocol can reduce the number of animals or specimens required in the experiment and is more economical for downstream analysis. However, it should be noted that some genes of microglia will be modulated by these inhibitors, which may limit the utilization of these inhibitors for future work24. Using the specialized dissociator instrument after the addition of enzyme mix 1 and enzyme mix 2 for tissue dissociation can also limit transcriptional responses in protocol step 323.
This protocol has some limitations. The purity of microglia is only approximately 85% in the protocol, which fails to comprise more than 90% of all single cells similar to other literatures19,22. The replacement of LS column with MS column, the absence of the filters, and the quality of microbeads may be the potential causes. If the purity of the isolated microglia fails to meet the requirements, prolonging the magnetic incubation time or passing the sorted cells through a second magnetic column will improve purity (Supplemental Figure S1). If the number of cells is low, tissue should be harvested from more mice and cell loss due to careless aspiration avoided. If the cell viability is low, replacing PBS in the loading buffer with RPMI 1640 medium could significantly improve the cell viability. It is also beneficial for cell viability to ensure that all the solutions used in the protocol are fresh, and the protocol is completed as soon as possible. It is important for users to balance the purity and the number of microglia obtained using this protocol.
The protocol utilizes the binding of microglia with CD11b beads and the enrichment in a magnetic field to purify the microglia. Therefore, purified microglia contain the contamination of myeloid cells, which is inevitable in this protocol. In contrast to FACS-the gold standard for sorting cells-the purity of microglia isolated by MACS is not sufficiently high for some elaborate applications such as deep sequencing, which limits its widespread usage. In addition, FACS for sorting microglia can eliminate the contamination of myeloid cells. However, MACS is a gentler method, retains more original morphological characteristics of glial cells especially astrocytes, takes less time for isolating microglia, and has a higher cell yield than FACS, suggesting that it can reduce the number of mice to be sacrificed and may be more suitable for time-sensitive experiments19,25. Meanwhile, MACS is more cost-effective and widely available and has lower technical requirements for experimenters. MACS has been proven the most reliable and consistent method to isolate microglia. Morphological analysis showed that microglia would not be activated by MACS, and RNA-seq has demonstrated that microglia isolated by this method maintain a resting state19,26.
Overall, the protocol is important for the survey of microglia in demyelinating diseases, and microglia isolated by this protocol can be utilized for various downstream applications such as quantitative RT-PCR and RNA-seq. The protocol utilizes neutral red dye to locate demyelinating lesions, which ensures the accuracy of lesion locations and reduces any confusion with normal tissue during sampling. Accurate localization of lesions can help focus on the effects of diseases and study the unique molecular phenotype and functional characteristics of microglia in demyelinating diseases. This will provide a basis for studying the mechanism of microglia in demyelinating diseases. This method of isolating microglia using magnetic CD11b beads can purify microglia with high cell viability and yield in a short time, with minimal requirements for experimental conditions, which allows widespread usage for studying microglia in different conditions.
The authors have nothing to disclose.
The study was supported by Tongji Hospital (HUST) Foundation for Excellent Young Scientist (Grant No. 2020YQ06).
1.5 mL Micro Centrifuge Tubes | BIOFIL | CFT001015 | |
15 mL Centrifuge Tubes | BIOFIL | CFT011150 | |
50 mL Centrifuge Tubes | BIOFIL | CFT011500 | |
70 µm Filter | Miltenyi Biotec | 130-095-823 | |
Adult Brain Dissociation Kit, mouse and rat | Miltenyi Biotec | 130-107-677 | |
C57BL/6J Mice | SJA Labs | ||
CD11b (Microglia) Beads, human and mouse | Miltenyi Biotec | 130-093-634 | |
Fetal Bovine Serum | BOSTER | PYG0001 | |
FlowJo | BD Biosciences | V10 | |
MACS MultiStand | Miltenyi Biotec | 130-042-303 | |
MiniMACS Separator | Miltenyi Biotec | 130-042-102 | |
MS columns | Miltenyi Biotec | 130-042-201 | |
Neutral Red | Sigma-Aldrich | 1013690025 | |
NovoCyte Flow Cytometer | Agilent | A system consisting of various parts | |
NovoExpress | Agilent | 1.4.1 | |
PBS | BOSTER | PYG0021 | |
Pentobarbital | Sigma-Aldrich | P-010 | |
Stereomicroscope | MshOt | MZ62 |