Flow Cytometry
Immune Cells |Granulocyte introduction and indicator selection
Source: Elabscience®Published: Oct 18,2024
Granulocytes are a type of white blood cell derived from hematopoietic stem cells in the bone marrow, characterized by the presence of specific granules in their cytoplasm. As innate immune cells, granulocytes play crucial roles in various diseases, including viral and parasitic infections, allergic reactions, chronic inflammation, asthma, immune regulation, autoimmune diseases, and cancer.
In this article, we will elaborate on granulocytes in detail, focusing on their classification, growth and development, and the selection of indicators for flow cytometric analysis.
Classification of Granulocytes
Granulocytes are categorized into four major types based on their morphology and function: neutrophils, eosinophils, basophils, and mast cells.
Neutrophils
Structural Features:
l Diameter approximately 10-15 μm.
l When immature, the nucleus is round and initially concave, and the cytoplasm is lighter in color and has nucleoli.
l After activation and maturity, it turns into a dark leafy nucleus with 3-5 lobes connected by a wisp of slender genetic material. The cytoplasm contains a large number of purple granules, called nitrogen-loving granules, and the nucleolus disappears.
Functions:
l First phagocytic cells to arrive at the site of inflammation.
l Primary and secondary granules in the cytoplasm contain a complex of various lysosomal enzymes, capable of phagocytosing and killing pathogens, as well as responding to DAMPs or PAMPs.
l Secrete cytokines to recruit other immune cells to the site of inflammation for immune regulation.
Abundance:
l Most abundant, comprising 50% to 70% of circulating white blood cells (approximately 20% in mice).
Eosinophils
Structural Features:
l Diameter approximately 10-16 μm.
l Nucleus is segmented or bilobed.
l Cytoplasmic granules contain specific cationic proteins.
Functions:
l Motile phagocytic cells originating from the bone marrow, with a short residence time in the bloodstream before migrating to tissues, where most reside.
l Provide host defense against parasitic infections and allergic diseases.
l Release lipids, peptides, and cytokines to mediate inflammation and host defense.
Abundance:
l Second in quantity, comprising 1-3% of circulating white blood cells, approximately 6% in the bone marrow.
Basophils
Structural Features:
l Diameter approximately 10-14 μm.
l Multilobed nucleus and prominent, brightly stained cytoplasmic granules.
Functions:
l Non-phagocytic granulocytes that induce immune responses against parasitic infections.
l Secrete histamine and cytokines, assisting in the modulation of adaptive immune responses, increasing microvascular permeability to facilitate leukocyte influx into inflamed tissues, and promoting Th2 immune responses.
Abundance:
l Basophils are the least numerous cells, comprising less than 1% of circulating white blood cells (approximately 0.5%).
Mast Cells
Structural Features:
l Diameter approximately 10-30 μm.
l Blurry boundaries of the nucleus and densely granular cytoplasm.
Functions:
l Secrete angiogenic factors to induce angiogenesis.
l Release newly synthesized mediators necessary for immune responses.
l Involved in allergic reactions.
Abundance:
l Mast cells do not reside in the bloodstream but are localized in various mucosal and epithelial tissues throughout the body, comprising less than 1% of circulating white blood cells.
Growth and Development of Granulocytes
The growth and development of granulocytes can be divided into six stages: myeloblasts, promyelocytes, myelocytes, metamyelocytes, band cells, and segmented neutrophils. Granulocytes are named for the presence of numerous granules in their cytoplasm. Granules first appear in type II myeloblasts, termed nonspecific granules (also known as A granules, or azurophilic granules). Specific granules (termed S granules) appear from the myelocyte stage onwards. There are three types of S granules: neutrophilic granules, eosinophilic granules, and basophilic granules. Based on the presence of specific granules, cells from the myelocyte stage onwards are classified into neutrophils, eosinophils, and basophils. The main differentiation characteristics and morphological differences are illustrated in the following figure:
Figure 1. Characteristics of Different Types of Granulocyte Differentiation and Morphological Differences
Selection of Indicators for Granulocyte Flow Cytometric Analysis
Differentiation and identification of granulocytes can be achieved based on surface markers. The main antibody selection differences between humans and mice are as follows:
Additionally, granulocytes in human peripheral blood exhibit higher side scatter (SSC) in flow cytometry analysis due to the specificity of their cytoplasmic granularity. This characteristic allows for their identification using FSC and SSC parameters on the flow cytometer, as illustrated in the figure below:
Figure 2. Staining of Human Peripheral Blood Cells with PerCP Anti-Human CD45 Antibody (E-AB-F1137F), followed by red blood cell lysis using 1× ACK Lysis Buffer (E-CK-A105) prior to flow cytometric analysis, allows for clear differentiation of granulocytes, monocytes, and lymphocyte populations.
Granulocyte analysis can also be performed using the granulocyte marker CD15 antibody, as illustrated in the figure below:
Figure 3. Staining of Human Peripheral Blood Granulocytes with PE Anti-Human CD15/SSEA-1 Antibody (E-AB-F1142D) (filled gray histogram), with unstained granulocytes (empty black histogram) as control.
The preceding text comprises all the information regarding granulocytes. For more introductions to immune cells, please continue to follow the "Flow Cytometry Classroom" series.
References:
[1] Mckenna, Ellen, et al. "Neutrophils: Need for Standardized Nomenclature." Frontiers in Immunology 12 (2021).
[2] Rose, Shawn, A. Misharin, and H. Perlman. "A novel Ly6C/Ly6G-based strategy to analyze the mouse splenic myeloid compartment." Cytometry Part A 81A (2012).
[3] Verde, de Peralta, Silvia S. "Color Atlas of Hematology. Practical Microscopic and Clinical Diagnosis, 2d ed." Journal of Pediatric Hematology/Oncology 26 (2004).
