Method Article

Identifying Microglia and Peripheral Infiltrating Macrophages in the Injured Spinal Cords Using Flow Cytometry

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DOI:

10.3791/67967

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June 24th, 2025

 ,  ,  ,  ,  ,  ,  , 
1Clinical Laboratory, the First Affiliated Hospital of Bengbu Medical University, 2Department of Biochemistry and Molecular Biology, School of Laboratory Medicine, Bengbu Medical University, 3Anhui Province Key Laboratory of Immunology in Chronic Diseases, Bengbu Medical University, 4The Second Affiliated Hospital of South China University, 5Anhui Province Key Laboratory of Basic and Translational Research of Inflammation-related Diseases, Bengbu Medical University

In This Article

Summary

Identifying microglia (MG) and peripheral infiltrating macrophages (Mø) in injured spinal cords is difficult. In this protocol, flow cytometry (FCM) was used to identify M1-like MG, M2-like MG, M1-like Mø, and M2-like Mø, respectively. This technology can also be applied to other central nervous system diseases to understand the role of MG and Mø.

Abstract

In a healthy spinal cord, peripheral infiltrating macrophages are almost undetectable, and microglia (MG) participate in maintaining the stability of the spinal cord microenvironment by phagocytosis, clearing cellular debris, and producing neurotrophic factors. After spinal cord injury (SCI), MG are activated, and peripheral immune cells infiltrate into the injured spinal cord. Among these immune cells, activated MG and peripheral infiltrating macrophages (Mø) play crucial roles in the pathological process of SCI. These cells can be distinguished into pro-inflammatory (M1-like) and anti-inflammatory (M2-like) phenotypes; however, distinguishing them is challenging due to their similarity in morphology and many cellular markers. Flow cytometry (FCM), a widely used technique in the biomedical field, can simultaneously detect multiple cellular parameters such as cell size, particle size, cell surface, and intracellular markers in a single experiment. Over years of research, FCM was attempted to identify MG and Mø in spinal cords and was continuously optimized to ultimately develop a stable detection method. This method allows for the identification of M1/M2-like MG and M1/M2-like Mø after SCI by detecting the expression profiles of CD45, CD11b, CD68, CCR7, and other markers. In this protocol, CD11b+CD45−/lowCD68+CCR7+, CD11b+CD45−/lowCD68+CCR7-, CD11b+CD45highCD68+CCR7+, and CD11b+CD45highCD68+CCR7- cells can be identified as M1-like MG, M2-like MG, M1-like Mø, and M2-like Mø, respectively.

Introduction

Spinal cord injury (SCI) is a result of spinal cord damage caused by various reasons. SCI has a high rate of disability, and currently, there are no effective treatment options, making it one of the most serious public health issues in the world1,2. The pathophysiological process of SCI is complex, divided into two stages. Initially, mechanical trauma from the injury immediately causes acute cellular dysfunction and cell death. Then, secondary damage leads to further cellular dysfunction and cell death over days, weeks, or even months. Within the secondary injury mechanisms, inflammation has been proven to be a key determinant of the severity of secondary damage, ultimately dictating cell death and cell functionality3. The characteristics of inflammatory responses are the infiltration and activation of inflammatory cells within the injured spinal cord, leading to an increase in inflammatory cells and inflammatory factors, creating an inflammatory microenvironment, and eventually causing spinal cord dysfunction4.

Microglia in the central nervous system (CNS) and peripheral infiltrating macrophages play a critical role in the inflammatory response following spinal cord injury (SCI)5,6. As resident macrophages of the CNS, microglia constitute 5%-10% of CNS cells and are essential for SCI recovery7,8. Microglia can engulf and remove cellular debris after SCI, mediating the formation of healing scars9,10. However, excessive activation of microglia can lead to a sustained inflammatory response and promote the formation of glial scars, which impede axonal regeneration11. After SCI, activated microglia mainly exhibit two phenotypes: M1-like and M2-like12,13. The M1-like phenotype is pro-inflammatory, promoting the synthesis of inflammatory factors contributing to cell apoptosis and secondary injury. M1-like microglia also have neurotoxic effects, exacerbating injury and neuronal apoptosis14,15. In contrast, the M2-like phenotype has anti-inflammatory properties and promotes angiogenesis, remyelination, axonal growth, and tissue repair16,17. M2-like microglia play a crucial role in modulating inflammation and repair processes.

Peripheral macrophages infiltrate into the lesion site after SCI through damaged blood-spinal cord barriers and vasculature17,18, further regulating the inflammatory response, phagocytosis, scar formation, and neural tissue regeneration19,20. Peripheral infiltrating macrophages can reduce the inflammatory response after SCI, phagocytose tissue debris21, promote neural regeneration, and matrix remodeling. However, a sustained inflammatory response mediated by peripheral infiltrating macrophages may lead to secondary injury, which is detrimental to long-term recovery22,23. M1-like peripheral infiltrating macrophages have strong phagocytic and antigen-presenting abilities, capable of clearing necrotic cells24. However, their excessive secretion of pro-inflammatory cytokines, reactive oxygen species (ROS), and reactive nitrogen species (RNS) can damage neurons and glial cells, leading to more severe neuronal apoptosis25. Conversely, M2-like peripheral infiltrating macrophages can inhibit neuronal apoptosis and mitigate the inflammatory response after SCI, thereby promoting the repair of neural tissue26.

Activated microglia and peripherally infiltrating macrophages after SCI exhibit morphological and molecular expression similarities that make them difficult to differentiate. Flow cytometry (FCM), a widely adopted technique, is employed to analyze the expression of cell surface and intracellular molecules, identify different cell subpopulations, and simultaneously assess multiple parameters of individual cells. A stable FCM assay has been pursued to distinguish activated microglia from peripherally infiltrating macrophages, facilitating the investigation of their roles in SCI. This article describes the stable detection method that has been developed.

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Protocol

This study was approved by the Animal Care Ethics Committee of Bengbu Medical University (No. 2020-050). Animal care complied with the regulations provided by the Ministry of Science and Technology of China. A total of 18 eight-week-old female C57/BL6 mice were commercially obtained and used. The details of the reagents and the equipment used are listed in the Table of Materials.

1. Experimental and animal preparation

  1. House the mice in ventilated cages before surgery. Maintain a 12-h light/dark cycle and provide free access to food and water. Keep the environmental room temperature at 20-22 °C and the humidity at 30%-70% for one week of acclimatization.
  2. Sterilize the surgical instruments for the mice by autoclaving and dry them before use. Subject the instruments and the animal surgery room to ultraviolet sterilization half an hour before surgery. Group and label the mice according to the random grouping principle. Record and register their weights for later use.
  3. Anesthetize the mice with a cocktail of ketamine (80 mg/kg) and xylazine (10 mg/kg) by intraperitoneal injection (following institutionally approved protocols). Shave the hair on the back from the T8 to T10 vertebrae.

2. Preparation of the SCI mouse model

  1. Disinfect the surgical area using an iodine swab from head to tail, centering on T9, followed by disinfection with a 75% alcohol swab. Make a 1 cm incision in the skin centered at T9. Dissect the fascia, muscle, and ligaments surrounding T9 for 1 cm.
    1. Detach the muscle adjacent to the T9 spinous process to expose T9. Use ophthalmic scissors to uncover and remove the lamina between T9 and T10, fully exposing the spinal cord located at T9, which is approximately 2 mm in diameter.
      NOTE: When lifting the T9 lamina, ensure that the scissors do not touch the spinal cord.
  2. Stabilize the spine by clamping the spinous processes of T7 and T11. Create a moderate SCI model using a rod with a diameter of 1.3 mm and a force of 0.5 N. Observe the tail curling upward and the hind limb twitching after SCI. For sham-operated mice, perform only laminectomy without contusion.
  3. Achieve hemostasis at the incision site. Perform layered closure using sterile 5-0 surgical sutures. For muscular approximation, grasp the deep fascia with Adson forceps with teeth and suture the panniculus carnosus layer using 5-0 absorbable monofilament, inserting the needle perpendicularly 2 mm from the wound edge to achieve symmetrical full-thickness bites (2-3 mm depth) spaced 3 mm apart.
    1. Tie square knots with controlled tension to align the fascial planes anatomically. Reposition the skin edges using micro Iris scissors to create 1 mm epidermal eversion. Align the dermis precisely with watchmaker's forceps and secure with simple interrupted 5-0 non-absorbable sutures spaced 2 mm apart, ensuring uniform coaptation without subcutaneous dead space.
    2. Disinfect the suture line with iodine swabs using unidirectional strokes from the wound center outward. Gently dab any residual blood with saline-moistened gauze and apply a thin layer of triple antibiotic ointment along the incision.
  4. Post-surgery, place the mice in a cage kept at a suitable temperature and humidity. Provide urination care twice daily (morning and evening) to assist the mice until they regain independent urination.
  5. Inject each mouse subcutaneously with 0.5 mL of saline and antibiotics (gentamicin, 2000-2500 U/mouse) daily to prevent infection.

3. Isolation of spinal cord tissue from C57/BL6 mice

  1. Anesthetize the mice before surgery (step 1.3). Once fully anesthetized, fix them on the surgical table in the supine position. Sham-operated mice are treated similarly.
  2. Make a transverse incision in the thoracic cavity, below the sternum, and along the diaphragm to expose the heart. Gently stabilize the heart with hemostats and insert the perfusion cannula into the apical left ventricle, advancing it into the aorta.
    1. Immediately cut the right auricle and begin perfusion at a rate of 250 mL/h with 10 mL of PBS solution. Perfusion is considered successful when the fluid draining from the right auricle turns from bloody red to clear and colorless, or when the liver transitions from red to white27.
  3. Transect the spine at the tail end of the mouse to expose the vertebral foramen. Using ophthalmic scissors, carefully dissect the spinal cord tissue 0.5 cm above and below the injury site. Place the isolated spinal cord tissue into a sterile, enzyme-free culture dish containing 5% staining buffer, and store it on ice at 4 °C for later use in colloidal silica-coated polyvinylpyrrolidone (PVP) density gradient centrifugation.
    NOTE: When dissecting the spinal cord near the injury center, ensure the integrity of the tissue to avoid tearing or damaging the spinal cord at the injury site. 5% Staining Buffer (SB) consists of 2.5 mL of fetal bovine serum (FBS) and 47.5 mL of phosphate-buffered saline (PBS).

4. Density gradient centrifugation using colloidal silica coated with polyvinylpyrrolidone

  1. Place the spinal cord tissue in a 300-mesh cell strainer and grind it using the plunger of a 5 mL/10 mL disposable syringe. After grinding, filter the cell suspension through the 300-mesh strainer into a 15 mL centrifuge tube for centrifugation (5 min, 110 x g, room temperature).
    1. After centrifugation, add 1 mL of trypsin cell digestion solution to the cell sediment, and incubate it in a cell culture incubator at 37 °C for 15 min. Stop the digestion by adding 1.5 mL of fetal bovine serum, then centrifuge again (110 x g, 5 min, room temperature). Resuspend the cell sediment in 1 mL of 5% staining buffer (SB).
  2. Sequentially add 4 mL of 70% colloidal silica-coated polyvinylpyrrolidone solution, 4 mL of 30% colloidal silica-coated polyvinylpyrrolidone solution, and 1 mL of the 5% SB-resuspended cell solution to a 15 mL centrifuge tube. Centrifuge the mixture (300 x g, 30 min, 20 °C).
    NOTE: Prepare the colloidal silica-coated polyvinylpyrrolidone separation reagent as a 9:1 mixture of colloidal silica-coated polyvinylpyrrolidone and 10× PBS. Dilute the 70% colloidal silica-coated polyvinylpyrrolidone solution with 1× PBS and the 30% colloidal silica-coated polyvinylpyrrolidone solution with DMEM or RPMI 1640 cell culture medium.
  3. After centrifugation, the cells will form layers in the centrifuge tube (Figure 1). Discard the cell debris and platelet layers above the 8 mL calibration mark. Carefully aspirate the remaining solution (up to the 1.5 mL calibration mark) and discard the sediment below the mark.
    1. Transfer the remaining solution to a new 15 mL centrifuge tube and add 1× PBS to bring the total volume to 15 mL for centrifugation (400 x g, 5 min, 4 °C).
  4. Discard the supernatant and resuspend the cell sediment in 5% SB. Transfer the solution to a flow cytometry (FCM) tube for washing and centrifugation (110 x g, 5 min, 4 °C). Resuspend the cell sediment in the FCM tube with 200 µL of 5% SB.

5. FCM staining

  1. Divide 200 µL of the cell suspension equally into two new flow cytometry tubes. Label one tube as "Isotype Control" and the other as "Specific Staining."
  2. Staining procedure
    1. To the "Isotype Control" tube, add the following isotype-matched antibodies: PE-labeled Rat IgG2b kappa, FITC-labeled Rat IgG2b kappa, APC-labeled Rat IgG2b kappa, and APC-eFluor 780-labeled Rat IgG2b kappa.
    2. To the "Specific Staining" tube, add the following panel of fluorescent-labeled antibodies according to the recommended dilution ratios: Rat anti-mouse CD45 APC, Rat anti-mouse CD11b PE, Rat anti-mouse CD68 FITC and Rat anti-mouse CCR7 APC-eFluor 780.
    3. Incubate the tubes at room temperature, protected from light, for 30 min.
  3. Wash the stained cells twice by adding 1 mL of SB each time. Centrifuge the cells (110 x g, 5 min, 4 °C). Finally, fix the cells with 2% paraformaldehyde (PFA) for flow cytometry analysis.

6. FCM detection

NOTE: For details of the procedure, refer to previous reports27,28.

  1. Analyze samples using the flow cytometer with the compatible software (see Table of Materials).
    1. First, analyze the isotype control tube to establish the primary graph (FSC on the x-axis and SSC on the y-axis). Adjust the forward scatter (FSC) and side scatter (SSC) voltages to position the cell population within an appropriate range.
    2. Set up two additional graphs: One with IgG-APC on the x-axis and IgG-PE on the y-axis. Another with IgG-FITC on the x-axis and IgG APC-Cy7 (note: APC-eFluor 780 was detected in this channel) on the y-axis.
    3. Adjust the fluorescence channel voltages for IgG-APC, IgG-PE, IgG-FITC, and IgG APC-Cy7 to position the cell population within the 103 of the double-negative region.
  2. Once the FSC and SSC voltages, as well as the fluorescence channel voltages, have been adjusted and optimized using the isotype control tube, these settings should remain unchanged. Proceed to analyze the antibody-stained experimental tubes.
    1. Establish a primary graph with CD11b-PE on the x-axis and SSC on the y-axis. Set up another graph with CD68-FITC on the x-axis and CCR7 APC-Cy7 on the y-axis. Using the isotype control tube as a reference, set the baseline values and delineate the "region" for CD11b+ cells. On the primary graph, gate the CD11b+ cell region and define it as the "P1 gate."
    2. Next, select the graph with CD68-FITC on the x-axis and CCR7 APC-Cy7 on the y-axis. Right-click and choose Show Population, then pick P1. This will display the cell populations within the CD11b+ group that express either CD68+/CCR7+ or CD68+/CCR7-. Collect 50,000 events per sample.
    3. After acquiring the data, adjust the fluorescence compensation as necessary. Analyze and determine the percentage of CD11b+ CD68+ CCR7+ (M1-like) cells and CD11b+ CD68+ CCR7- (M2-like) cells. The average percentage of positively marked cells is expressed as a percentage of the total sample population.
  3. Using the well-adjusted voltage parameters from the isotype control tube, proceed with the analysis of the experimental tubes.
    1. First, establish a graph with CD45-APC and CD11b-PE, followed by two graphs with CD68-FITC and CCR7 APC-Cy7. Collect 50,000 events per sample, and after acquiring the data, adjust the fluorescence compensation.
    2. In the CD45/CD11b pseudocolor plots, analyze the "regions" for CD11b+ CD45−/low and CD11b+ CD45high cells for each sample. Then, analyze the CD68+ CCR7+ cells and CD68+ CCR7- cells within these two regions.
    3. Integrate the analyses to determine the percentages of the following cell populations: CD11b+ CD45−/low CD68+ CCR7+ (M1-like microglia), CD11b+ CD45−/low CD68+ CCR7- (M2-like microglia), CD11b+ CD45high CD68+ CCR7+ (M1-like macrophages), CD11b+ CD45high CD68+ CCR7- (M2-like macrophages).
      NOTE: The average percentage of positively marked cells is expressed as a percentage of the total sample population.

7. FCM data analysis

  1. Launch the flow cytometry analysis software and drag the flow cytometry experiment files to the "All Samples" section on the interface. The flow cytometry experiments will be displayed below "All Samples" on the interface. Begin by analyzing the isotype control tube to determine the threshold between positive and negative fluorescence signals.
    1. Double-click on the isotype control tube sample to bring up a graphical window displaying a two-dimensional dot plot. Select FSC-A for the x-axis and SSC-A for the y-axis. Click on the Rectangular Gate button at the top of the interface and use the rectangle to select the area after removing cellular debris (Figure 2A).
    2. Double-click on the selected area to generate a new graphical window. Choose FITC for the x-axis and Histogram for the y-axis. Click on the Region Gate button at the top of the interface to set a gate in the single-parameter histogram to determine the threshold between negative and positive fluorescence signals. The region gate will define the positive area (Figure 2A).
      NOTE: The area with smaller FSC values represents cellular debris.
    3. For each of the four fluorescent antibodies used in the experiment, determine the threshold between negative and positive signals using the isotype control tube sample. On the x-axis, sequentially change the fluorochromes to PE, APC, and APC-Cy7, while keeping the y-axis as Histogram.
    4. Use the region gate tool in the single-parameter histogram to set gates and determine the threshold between negative and positive signals for each of the four fluorescent antibodies (Figure 2A).
  2. To analyze the proportions of M1-like and M2-like cells:
    1. Double-click on the experimental tube sample to bring up a two-dimensional dot plot. Select FSC-A for the x-axis and SSC-A for the y-axis. Click on the Rectangular Gate button at the top of the interface and use the rectangle to select the area after removing cellular debris (Figure 2B).
    2. Double-click on the selected area to generate a new graphical window. Choose CD11b-PE for the x-axis and SSC-A for the y-axis. Click on the Rectangular Gate button at the top of the interface and, using the PE positive/negative threshold set by the isotype control as a reference, define the CD11b+ gate (Figure 2B).
    3. Double-click on the CD11b+ gate area to generate a new graphical window. Select CD68-FITC for the x-axis and CCR7 APC-Cy7 for the y-axis. Click on the Cross Gate button. Using the FITC and APC-Cy7 positive/negative thresholds set by the isotype control as references, use the cross gate to categorize the cells, determining the proportions of CD11b+ CD68+ CCR7+ (M1-like) cells and CD11b+ CD68+ CCR7- (M2-like) cells (Figure 2B).
      NOTE: The gating principles for analyzing the proportions of M1-like macrophages (M1-like Mø), M2-like macrophages (M2-like Mø), M1-like microglia (M1-like MG), and M2-like microglia (M2-like MG) are consistent with those used for analyzing M1-like and M2-like cells. The thresholds for distinguishing positive and negative fluorescence signals are based on the isotype control tube, which serves as the reference for setting the gates. The software operation process is illustrated in Figure 2B, and the marker expression profiles used for determining the cell proportions are described in detail in the representative results section.

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Results

The immune cells from spinal cord tissue are separated using colloidal silica coated with polyvinylpyrrolidone density gradient centrifugation. During the centrifugation process, cells are distributed into different layers according to their buoyant densities, thereby achieving cell separation (Figure 1). From top to bottom, the distribution is as follows: cellular debris, mononuclear cell-rich layer, lymphocyte-rich layer, and at the bottom, the erythrocyte and granulocyte layer.

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Discussion

The successful creation of an SCI animal model is essential. The SCI model was established by exposing the spinal cord at the T9 segment to a force of 0.5 N. The success of the animal model was demonstrated by the mouse's tail curling up, both hindlimbs experiencing spasms, and the hindlimbs being paralyzed, making it impossible for the mouse to walk after the SCI (see step 2.3). Postoperative care is critical for the survival of the mouse. Urinary care is provided until the mouse can urinate independently. It is als...

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Disclosures

No conflicts of interest were declared.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 82072416); and the high-level scientific and technological innovation team fund of the First Affiliated Hospital of Bengbu Medical College (BYYFY2022TD001).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
1×PBS SolarbioP1020
10×PBS SolarbioP1022
18 eight-weeks-old female C57/BL6 mice Cavens Laboratory Animal Ltd, Chang Zhou, China
APC-eFluor 780-labeled Rat anti-mouse CCR7 (IgG2b kappa)Invitrogen47-1971-820.25μg/test
APC-eFluor 780-labeled Rat IgG2b kappa  isotype controlInvitrogen47-4321-820.25μg/test
APC-labeled Rat anti-mouse CD45 (IgG2b kappa)Invitrogen17-0451-820.125μg/test
APC-labeled Rat IgG2b kappa  isotype controlInvitrogen17-4031-820.125μg/test
DMEMGibco11965092
Fetal bovine serumLonseraS711-001
FITC-labeled Rat anti-mouse CD68 (IgG2b kappa)Invitrogen MA5-166760.25μg/test
FITC-labeled Rat IgG2b kappa  isotype controlInvitrogen11-4031-820.25μg/test
Paraformaldehydebiosharp23319084
PE-labeled Rat anti-mouse CD11b (IgG2b kappa)Invitrogen12-0112-810.25μg/test
PE-labeled Rat IgG2b kappa  isotype controlInvitrogen12-4031-820.25μg/test
Percoll SolarbioP8370
Trypsin SolutionbiosharpBL526A

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Microglia IdentificationSpinal Cord InjuryDensity Gradient CentrifugationImmune Cell IsolationCD11b MarkerCD68 MarkerCCR7 MarkerM1 M2 Phenotypes

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