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Granulocytes

1 Introduction

Granulocytes are myeloid cells that play key roles in innate immune responses. They are characterized by the presence of cytoplasmic granules and a poly-lobed nucleus. Therefore, granulocytes are also termed polymorphonuclear leukocytes (PMN, PML, or PMNL).

Generated and differentiated in bone marrow, granulocytes have a limited life span once in the periphery, and thus cannot be maintained in culture for prolonged periods of time. They play a well-known role in inflammation and allergy, but are also capable of antigen presentation and immune response modulation (PMID: 23007469). Among the first responders at sites of inflammation, granulocytes are equipped with pre-formed granules filled with an array of potent effector molecules to fight invading pathogens. Interestingly, depending on their activation status and environment, granulocytes have also been implicated in disease. For example, neutrophils can promote tumor growth, modulate their extracellular matrix, or enhance tumor angiogenesis (PMID: 26819959, 23485549, 28099862, 27558343).

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Dendritic cells (mouse)

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2 Granulocytes in various tissues

Granulocytes circulate through the body via peripheral blood and can be found in numerous peripheral tissues. However, they are present in such small numbers that many studies are performed on granulocytes derived from progenitor cells. Miltenyi Biotec provides optimized products to isolate primary granulocytes from various tissues, as well as standardized instruments and reagents to generate granulocytes from progenitor cells.

2.1 Cell subset, frequencies, and marker expression

At a glance: granulocyte subtypes

Cell subtypeFrequencyMarkersFunction
NeutrophilsFrequencies of granulocytes are variable and depend on tissue and mouse strainCD11b, Ly-6G, Gr-1, CD115–• Destruction of pathogens by phagocytosis and release of antimicrobials 
• Recruitment of other immune cells
EosinophilsCD11b, Ly-6C, Siglec-F, CD193 (CCR3)• Effector cells in allergic inflammation and responses against pathogens 
• Active role in diseases associated with eosinphilia (asthma, skin diseases, etc.).
BasophilsCD11b, CD49b, FcεRIα, CD200R3, CD117–• Release of mediators that promote blood flow to site of infection 
• Promotion of TH2 differentiation 
• Release of cytokines

Granulocytes are divided into three major subtypes: neutrophils, eosinophils, and basophils. Granulocyte numbers vary greatly depending on tissue type and mouse strain, but granulocytes account for approximately 5–9% of peripheral blood cells and 1–2% of spleen cells in mice.

2.2 Sample preparation of tissues

In general, granulocytes can be isolated directly from peripheral blood without prior sample preparation such as the generation of PBMCs. Other tissues must be dissociated gently to create a single-cell suspension for downstream applications, including isolation of cell subpopulations, cell culture, or flow cytometry analysis. For details on sample preparation, see related chapters.

MACS Handbook:

Sample preparation

Spleen (mouse)

Lymph node (mouse)

2.3 Miltenyi Biotec application protocols for granulocytes from lymphoid tissue

Miltenyi Biotec has created dedicated application protocols for working with mouse granulocytes.

2.4 Magnetic separation of granulocytes from various tissues

Miltenyi Biotec has developed numerous products for the magnetic separation of granulocytes. Both positive selection and depletion strategies can be used to isolate granulocytes. Depletion is the preferred choice when working with bone marrow, whereas positive selection is most appropriate for other tissues.

For details on MACS® Cell Separation Technology, see the MACS Handbook chapter Magnetic cell separation .

MACS Handbook:

Magnetic cell separation

2.4.1 Isolation of neutrophils from various tissues

At a glance: Kits and reagents for the separation of neutrophils

Cell subtypeStarting materialIsolation strategyCommentsAutomationProduct
NeutrophilsBone marrow, lung, and bloodPositive selection of target cells Yes*Anti-Ly-6G MicroBeads UltraPure, mouse
NeutrophilsBone marrow or blood cell suspensionsDepletion of non-target cellsUntouched neutrophilsYes*Neutrophil Isolation Kit, mouse
Neutrophils and eosinophilsBlood and other tissuesPositive selection of target cells Yes*Siglec-F MicroBeads, mouse
*Automation options range from fully automated benchtop solutions such as the autoMACS® Pro Separator to high-throughput platforms such as the MultiMACS™ Cell24 Separator Plus or MultiMACS X.

Ly-6G, a murine lymphocyte antigen 6/urokinase-type plasminogen activator receptor (LU) family member with no human ortholog, is commonly used to identify mouse neutrophils (PMID: 17884993). Expression of Ly-6G is not restricted to neutrophils. Some eosinophils and granulocytic myeloid-derived suppressor cells (G-MDSC) express it at low levels in various diseases. Miltenyi Biotec offers the Anti-Ly-6G MicroBeads UltraPure, mouse for positive selection or depletion of mouse neutrophils from single-cell suspensions of mouse bone marrow, lung, and blood.

Human neutrophils can be easily harvested in large numbers from blood (PMID: 18432834). In contrast, the small volume of mouse blood makes it rather difficult to isolate sufficient numbers of neutrophils to perform downstream analyses. Bone marrow is a convenient alternative source of neutrophils and their precursors, both at steady-state under homeostatic conditions and during activation under a variety of infectious and non-infectious inflammatory conditions. Bone marrow neutrophils are functionally similar to blood neutrophils in mice, are reported to survive longer in culture (PMID: 14694182), and can become activated. The number of cells that can be obtained is compatible with the majority of immunological assays and adoptive transfer studies, making bone marrow a useful source for isolating neutrophils in a variety of mouse strains and disease models.

The Neutrophil Isolation Kit, mouse enables isolation of untouched neutrophils from bone marrow. Non-target cells are labeled with a cocktail of biotinylated antibodies for subsequent depletion, resulting in a highly pure population of isolated neutrophils, which are viable and ready for functional studies (PMID: 28783679). This kit can also be used to isolate neutrophils from blood cell suspensions.

Neutrophil Isolation Kit, mouse
Unseparated fraction
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Isolated neutrophils
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Murine neutrophils isolated from bone marrow. Neutrophils were isolated from C57BL/6 bone marrow cells using the Neutrophil Isolation Kit, mouse, an LS Column, and a MidiMACS™ Separator. Cells were fluorescently stained with Anti-Ly-6G-PE and CD11b-FITC. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

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2.4.2 Isolation of eosinophils from various tissue

At a glance: Kits and reagents for the separation of eosinophils

Cell subtypeStarting materialIsolation strategyCommentsAutomationProduct
Neutrophils and eosinophilsBlood and other tissuesPositive selection of target cellsYes*Anti-Siglec-F MicroBeads, mouse
*Automation options range from fully automated benchtop solutions such as the autoMACS® Pro Separator to high-throughput platforms such as the MultiMACS™ Cell24 Separator Plus or MultiMACS X.

Eosinophil-deficient mouse strains, such as Delta dbIGATA (PMID: 17641035) or MBP-1/EPX knockout mice (PMID: 23736699), can be useful systems to study eosinophilic disorders. In addition, eosinophils isolated from tissues are an important option to study the function of these cells. In most tissues murine eosinophils are identified based on the marker combination of Siglec-F and CD193 (CCR3) (PMID: 19178537). Miltenyi Biotec offers Anti-Siglec-F MicroBeads, mouse to enable isolation of eosinophils from various tissues. It is important to note that the cell populations isolated by these MicroBeads are a mixture of myeloid cells, the composition of which depends on the tissue of origin. Isolation of Siglec-F+ cells from blood leads to highly enriched eosinophils, whereas separation from lung cell suspensions results in cell populations containing both eosinophils and alveolar macrophages.

Anti-Siglec-F MicroBeads, mouse

Blood

Before separation
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Siglec-F+ cells
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Anti-Siglec-F MicroBeads, mouse

Lung

Before separation
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Siglec-F+ cells
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Eosinophils isolated from mouse blood and mouse lung tissue. Anti-Siglec-F MicroBeads, two MS Columns, and a MiniMACS™ Separator were used to isolate eosinophils from mouse blood and mouse lung. Isolation from blood resulted in highly enriched eosinophils, which were stained with Anti-Siglec-F-APC and Anti-Gr-1-VioGreen™ for flow cytometry analysis. Isolation from lung resulted in a mixture of eosinophils and Siglec-F+ alveolar macrophages. These cells were stained with Anti-Siglec-F-APC and CD11c-PE-Vio® 770. Analyses were performed on the MACSQuant® Analyzer. Cell debris and dead cells were excluded form the analysis based on scatter signals and propidium iodide fluorescence. Equal event numbers are shown for the respective dot plots.

2.4.3 Generating eosinophils by culturing bone marrow

At a glance: Kits and reagents for culturing bone marrow to generate eosinophils

Use Comments Product
Supplement Expansion of progenitor cells Mouse SCF
Supplement Mouse Flt3-Ligand
Supplement Mouse IL-5

The low frequency of eosinophils in mouse tissue has hampered research on these cells, but cell culture protocols developed by Helen Rosenberg (PMID: 20228959) help overcome this drawback. Starting from complete murine bone marrow, a cell suspension of pure eosinophils can be obtained in a 13-day process using SCF, Flt3-Ligand and IL-5.

MACS Handbook:

Cell culture

2.5 Characterization of granulocytes by flow cytometry

Granulocytes can be analyzed by flow cytometry using intracellular and surface staining.


 

At a glance: Markers for the discrimination of granulocyte subtypes (PMID: 26793192)

 Surface or intracellular markers
 CD11bCD49bFcεRIαCD177Ly-6GLy-6CCD115Siglec-FCD200R3
Neutrophils+–––+Lo–––
Eosinophils+––––Lo–+–
BasophilsGradually indistinct population, going from dim to positive ++–––––+
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