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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, which gives them the name 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 site of inflammation, granulocytes are equipped with preformed 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).
MACS Handbook:
Dendritic cells (mouse)
Related PDFs:
Granulocyte research (brochure)
At a glance: granulocyte subtypes
| Cell subtype | Frequency | Markers | Function |
| Neutrophils | Frequencies of granulocytes are variable and depend on tissue and mouse strain | CD11b, Ly-6G, Gr-1, CD115- |
|
| Eosinophils | CD11b, Ly-6C, Siglec-F, CD193 (CCR3) |
| |
| Basophils | CD11b, CD49b, FcεRIα, CD200R3; CD117- |
|
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 preferred when working with bone marrow, and positive selection when working with other tissues.
For details on MACS® Cell Separation Technology, see the MACS Handbook chapter Magnetic Cell Separation . MACS Handbook:
Magnetic cell separation
At a glance: Kits and reagents for the separation of neutrophils
| Cell subtype | Starting material | Isolation strategy | Comments | Automation | Product |
|---|---|---|---|---|---|
| Neutrophils | Lymphoid tissues | Positive selection of target cells | Yes* | Anti-Ly-6G MicroBead Kit, mouse | |
| Neutrophils | Bone marrow or blood cell suspensions | Depletion of non-target cells | Untouched neutrophils | Yes* | Neutrophil Isolation Kit, mouse |
| Neutrophils and eosinophils | Blood and other tissues | Positive 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. | |||||
In contrast to human neutrophils which are easily harvested in large numbers from blood (PMID: 18432834), the small volume of mouse blood makes isolating sufficient numbers of neutrophils to perform downstream analyses rather difficult. 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 reservoir 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.
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.
Related PDFs:
MACS Cell Separation - Select the best (brochure)
Granulocyte research (brochure)
Related videos:
At a glance: Kits and reagents for the separation of eosinophils
| Cell subtype | Starting material | Isolation strategy | Comments | Automation | Product |
|---|---|---|---|---|---|
| Neutrophils and eosinophils | Blood and other tissues | Positive selection of target cells | Yes* | 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. | |||||
Blood
Lung
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 purely enriched eosinophils, which were stained with Anti-Siglec-F-APC and 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-PE and CD11c-PE-Vio770 for flow cytometry analysis. Analyses were performed on the MACSQuant® Analyzer, and cell debris and dead cells were excluded based on scatter signals and propidium iodide fluorescence. Equal event numbers are shown for the respective dot plots.
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 |
MACS Handbook:
Cell culture
At a glance: Markers for the discrimination of granulocyte subtypes (PMID: 26793192)
| Surface or intracellular markers | |||||||||
| CD11b | CD49b | FcεRIα | CD177 | Ly-6G | Ly-6C | CD115 | Siglec-F | CD200R3 | |
| Neutrophils | + | – | – | – | + | Lo | – | – | – |
| Eosinophils | + | – | – | – | – | Lo | – | + | – |
| Basophils | Population smear gradually going from dim to positive | + | + | – | – | – | – | – | + |
Related PDFs:
REAfinity Recombinant Antibodies (brochure)
Granulocyte research (brochure)
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