MACS Handbook

Granulocytes

1 Introduction

Granulocytes belong to the myeloid cell family and represent the most abundant cell type in peripheral blood. 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). Special staining techniques developed by Paul Ehrlich in the late 1800s allowed the identification of granulocytes in blood, and opened the possibility to study these cells in different pathological conditions.

Granulocytes are derived from stem cells in bone marrow. They have a short life span in the periphery that can be prolonged at sites of infection. Located at strategic points in the body with preformed cytoplasmic granules filled with various effector molecules, they are among the first responders at the onset of innate immune responses. Granulocytes play a well-known role in inflammation and allergy, but are also capable of antigen presentation and immune response modulation (PMID: 23007469). The toxic mediators produced by these cells have been undeniably linked to various pathologies, and therapeutic approaches targeting granulocytes are currently under development to treat allergic diseases and asthma (PMID: 28283697, 26819959, 23485549, 28099862, 27558343).

2 Granulocytes in peripheral blood

Granulocytes circulate through the body via peripheral blood and account for approximately 25–75% of CD45+ leukocytes. They can be divided into three subtypes – neutrophils, eosinophils and basophils – named according to their characteristics staining with hematoxylin and eosin. Basophils stain dark blue, eosinophils red, and neutrophils stain a middle-ground pink.

2.1 Cell subsets, frequencies, and marker expression

At a glance: Granulocyte subtypes in peripheral blood
Cell subtypeFrequencyMarkersFunction
Neutrophils25–75% of total circulating human white blood cellsCD15, CD16, FcεRIα, CD66b, CD11b, CD49d
  • Destroys pathogen by phagocytosis and release of anti-microbials
  • Recruits other immune cells
Eosinophils1–10% of peripheral leukocytesCD15, CD66b, CD11b, Siglec8, CD193, CD16
  • Release of mediator molecules upon activation
  • Involved in antigen presentation
  • Polarize T cells
Basophils0.2–1% of white blood cellsCD15, FcεRIα, CD123, CD11b, CD203c, CD117
  • Release of mediators that promote blood flow to site of infection
  • Promote TH2 differentiation
  • Release cytokines

Neutrophils represent the most abundant type of granulocytes, accounting for 25–75% of all circulating human white blood cells (PMID: 23435331, 22491176). Their mechanisms of action encompass degranulation of antimicrobial proteins, release of chemotactic factors (PMID: 22392929, 20140197, 25512469, 21555529), and formation of neutrophil extracellular traps or NETs (PMID: 23435331, 22491176, 15001782) in response to pathogens. Typical markers are CD15, CD66b, CD16, and absence of CD49d.

Representing 1–10% of peripheral leukocytes, eosinophils in resting state reside mainly in the periphery, especially in lamina propia (PMID: 11877470, 25049430). They readily release granule content including eosinophil cationic protein (ECP), eosinophil peroxidase (EPX) and major basic protein (MBP), chemokines, and cytokines in response to pathogens (PMID: 25003763), and they play a role in antigen presentation and T cell polarization (PMID: 25003763, 8450230, 20065995, 16551246). A specific marker of eosinophils in human blood is Siglec8. Other commonly used markers are CD15, CD193, and absence of CD16.

Basophils are a small population, accounting for only 0.2–1% of white blood cells (PMID: 28506528). They are rich in preformed granules containing histamine, leukotrienes (LTs), and platelet-activating factor (PAF). Under specific conditions, they play non-redundant roles as effector cells and promote TH2 differentiation. They can release high amounts of IL-4 and may take part in dendritic cell modulation. A specific marker for blood Basophils is CD203c. Other typical markers are FcεRIα, CD123, and absence of CD117 (PMID: 27135604, 23558889).

2.2 Sample preparation of pheripheral blood

Eosinophils and neutrophils may be isolated directly from whole blood, requiring no upfront sample preparation. For some applications and for the isolation of basophils, blood, buffy coat, or buffy cone are first processed to generate PBMCs. For additional information about human peripheral blood, see Human peripheral blood. à link to Detail page Source: Human – peripheral blood

Miltenyi Biotec has developed a special protocol to generate PBMCs from whole blood for subsequent isolation of granulocytes. This protocol provides step-by-step instructions for performing a Percoll™ gradient on freshly drawn human blood treated with an anticoagulant (preferably EDTA or citrate, ACD-A, CPD) or fresh (not older than 8 hours) defibrinated blood or buffy coat treated with an anti-coagulant. The white blood cell layer directly above the red blood cells is collected and diluted with buffer to wash the cells. Any remaining red blood cells are then lysed and the white blood cells are washed twice. Finally, the cell pellet is resuspended in an appropriate buffer, and cells are counted before proceeding with magnetic cell separation. This protocol can be found as an appendix in data sheet of the CD16 MicroBeads, human, which you can download from the Related Resources panel to the right.
MACS Handbook:

Blood (human)

Related documents:

CD16 MicroBeads, human (data sheet)

2.3 Magnetic separation of granulocytes from peripheral blood

Miltenyi Biotec has developed numerous products for the straightforward magnetic separation of granulocyte subsets. Both positive selection and depletion strategies can be pursued for isolation directly from blood. Several positive selection strategies are possible to isolate granulocytes from PBMCs.

For details on MACS® Cell Separation Technology, see the MACS handbook chapter Magnetic cell separation
2.3.1 Isolation of neutrophils and eosinophils from whole blood
At a glance: Kits and reagents for the separation of neutrophils and eosinophils from whole blood
Cell subtypeStarting materialIsolation strategyCommentsAutomationProduct
Eosinophils and neutrophilsWhole bloodPositive selection of target cellsRecognizes 3-FAL, also designated Lewis X. Antigen not expressed on basophils and lymphocytes.

Yes*

StraightFrom Whole Blood CD15 MicroBeads, human
Eosinophils and neutrophilsWhole bloodPositive selection of target cellsAntigen expressed on eosinophils and neutrophils, but not basophils and lymphocytes.

Yes*

StraightFrom Whole Blood CD66b MicroBeads, human
EosinophilsWhole bloodDepletion of non-target cellsIsolates untouched eosinophils from up to 30 mL anticoagulated whole blood.

No

MACSxpress Eosinophil Isolation Kit, human
NeutrophilsWhole bloodDepletion of non-target cellsIsolates untouched neutrophils from up to 30 mL anticoagulated whole blood.

No

MACSxpress Neutrophil Isolation Kit, human
*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

StraightFrom™ MicroBeads can be used to isolate granulocytes directly from whole blood. Pre-enrichment of white blood cells by density gradient centrifugation is not required. The purified cells are well-suited for further flow cytometry analysis, molecular biology studies, and functional studies.

MACSxpress® Technology is a cell separation platform especially designed for processing large volumes of anticoagulated whole blood. With MACSxpress Technology, untouched human leukocytes are rapidly isolated from up to 30 mL of anticoagulated whole blood by removing immunomagnetically labeled non-target cells. The MACSxpress Eosinophil Isolation Kit, human and MACSxpress Neutrophil Isolation Kit, human have been tested by independent labs and found to be the optimal solutions compared to density gradient and competitor products (PMID: 28647456, 27567327).
MACSxpress Neutrophil Isolation Kit, human 
Unseparated fraction
After separation

Efficient enrichment of neutrophils from whole blood. Untouched neutrophils were isolated from 8 mL of human EDTA anticoagulated whole blood using the MACSxpress® Neutrophil Isolation Kit, a MACSmix Tube Rotator, and a MACSxpress Separator. The isolated cells were fluorescently stained with CD14-PerCP (# 130-094-969), CD15-PE (# 130-091-375), CD16-APC (# 130-091-246), CD45-VioBlue (# 130-092-880), and CD193-FITC (# 130-097-064) and analyzed by flow cytometry using the MACSQuant® Analyzer. Cell debris, non-leukocytes, and dead cells were excluded from the analysis based on CD45 expression, scatter signals, and propidium iodide fluorescence.

Noteworthy is that neutrophils are extremely susceptible to non-specific activation. Analysis of surface expression markers, adhesion, migration, phagocytic capacities and reactive oxygen species (ROS) production demonstrate that isolation with the MACSxpress Neutrophil Isolation Kit, human does not activate neutrophils. A scientific poster describing results from this analysis can be downloaded from the Related Resources panel to the right.
Related videos:
2.3.2 Isolation of basophils and eosinophils from PBMCs
At a glance: Kits and reagents for the separation of basophils and eosinophils from PBMCs
Cell subtypeStarting materialIsolation strategyCommentsAutomationProduct
BasophilsPBMCsDepletion of non-target cellsUses indirect magnetic labeling to remove non-target cells and achieve high-purity basophil isolates

Yes*

Basophil Isolation Kit II, human
BasophilsPBMCsPositive selection of target cellsConcurrent isolation of basophils and plasmacytoid dendritic cells

Yes*

CD123 MicroBeads, human
EosinophilsPBMCsDepletion of non-target cellsUses indirect magnetic labeling system to isolate untouched eosinophils

Yes*

Eosinophil Isolation Kit, human
*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

Both positive selection and depletion strategies are possible with different Miltenyi Biotec products. The Basophil Isolation Kit II, human and Eosinophil Isolation Kit, human use a cocktail of biotin-conjugated antibodies to indirectly label non-target cells. The highly pure eosinophils or basophils that result from the depletion of the magnetically labeled cells are suitable for functional studies on signal transduction, activation mechanisms, or cytokine production.

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Working scheme for the isolation of untouched eosinophils using the Eosinophil Isolation Kit, human.

Eosinophil Isolation Kit, human
Unseparated fraction
Enriched eosinophils

High purity of isolated eosinophil population. PBMCs generated from human peripheral blood served as starting material to isolate untouched eosinophils using the Eosinophil Isolation Kit, human, an LS Column, and a MidiMACS™ Separator. Cells were fluorescently stained with CD16-FITC. Eosinophils do not express CD16.

Related PDFs:
Related videos:

2.4 Characterization of granulocytes by flow cytometry

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Flow cytometry analysis of whole blood after erythrocyte lysis. (A) Scatter profile of all white blood cells after dead cell and debris exclusion. Neutrophils can be identified based on high granularity (SSC) and CD16 receptor expression, whereas eosinophils lack CD16. (B) Scatter profile of back-gated neutrophils and eosinophils. (C) Basophil-gating after dead cell and debris exclusion. Basophils are defined as CD203c+CD123+ cells.