Granulocytes

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 the absence of CD49d.

Representing 1–10% of peripheral leukocytes, eosinophils in resting state reside mainly in the periphery, especially in lamina propria (PMID: 11877470, 25049430). They readily release their granule content including eosinophil cationic protein (ECP), eosinophil peroxidase (EPX), and major basic protein (MBP) as well as chemokines, and cytokines in response to pathogens (PMID: 25003763). 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 Siglec-8. Other commonly used markers are CD15, CD193, and the absence of CD16.

Basophils are a small population, accounting for only 0.2–1% of white blood cells (PMID: 28506528). They are rich in pre-formed granules containing histamine, leukotrienes (LTs), and platelet-activating factor (PAF). Under specific conditions, they play non-redundant roles as effector cells and promote T_H2 differentiation. They can release large 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 the absence of CD117 (PMID: 27135604, 23558889).

Eosinophils and neutrophils can be isolated directly from whole blood, without prior 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 the MACS Handbook chapter Human cell sources – Blood (human). 

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 density gradient using freshly drawn human blood treated with an anticoagulant (preferably EDTA or citrate, ACD-A, CPD) or fresh defibrinated blood or buffy coat (not older than 8 hours) treated with an anticoagulant. The resulting 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 the data sheet of the CD16 MicroBeads, human, which you can download from the related resources panel to the right.

Miltenyi Biotec has developed numerous products for the straightforward magnetic separation of granulocyte subsets. Both positive selection and depletion strategies can be pursued for cell 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. 

Granulocytes can be analyzed by flow cytometry using intracellular and surface staining. Markers commonly used to analyze neutrophils, basophils, and eosinophils are listed in Cell subsets, frequencies, and marker expression.

Like many of the myeloid cell types, granulocytes are quite sticky. Thus, we recommend using REAfinity™ Antibodies whenever possible, to achieve optimal staining for flow cytometry. These recombinantly engineered antibodies are highly specific and require no FcR blocking as the Fc region is specifically mutated to abolish FcγR binding. Therefore, these antibodies are especially well-suited to stain myeloid cells. For more information about Miltenyi Biotec antibodies, see Cell analysis reagents – antibodies.

A common problem encountered by researchers studying myeloid cells is the sporadic appearance of markers used to identify and potentially isolate distinct cell subtypes. Granulocytes are no exception in this respect. In general, identification by flow cytometry can be done based on side scatter (SSC) and forward scatter (FSC) characteristics for the most abundant neutrophils and eosinophils, because they are highly granular and thus, segregate well from lymphocytes and monocytes. This is not true for basophils. Identification based on the expression of a single specific antigen is restricted to eosinophils (Siglec-8⁺) and basophils (CD203c⁺) in blood, whereas neutrophils are commonly identified via marker combinations (CD15⁺CD16⁺).