Neutrophils are polymorphonuclear leukocytes that serve as the first responders in the innate immune system. These cells rapidly migrate from the bloodstream to sites of infection or tissue damage, where they neutralize pathogens through phagocytosis, degranulation, and the release of Neutrophil Extracellular Traps (NETs). Because human studies are challenging, mouse models are the primary tool for scientists to dissect neutrophil functions in health and disease. Identifying and isolating these cells from various tissues, such as the bone marrow, blood, or inflamed organs, requires the precise use of surface markers. These markers act like molecular barcodes, allowing researchers to distinguish neutrophils from other immune cell types and track their behavior.
Essential Surface Markers for Identification
The definitive identification of mouse neutrophils relies on the co-expression of two primary surface proteins: CD11b and Ly6G. This dual-marker approach is consistently used across nearly all experimental systems to establish the basic neutrophil population. CD11b (Integrin alpha M or Mac-1) is a subunit of an adhesion molecule that enables neutrophils to stick to the inner lining of blood vessels and migrate into tissues. Its function is directly tied to the cell’s ability to respond to inflammatory signals.
CD11b expression alone is insufficient to define a neutrophil, as this protein is also found on other myeloid cells, including macrophages, monocytes, and dendritic cells. The essential counter-marker for specificity is Ly6G, a protein that is part of the Lymphocyte Antigen 6 complex. Ly6G is uniquely and highly expressed on mature mouse neutrophils, making it the most reliable single marker for this lineage in peripheral blood and tissue. Researchers often prefer the antibody clone 1A8 because it specifically binds to Ly6G.
Ly6G is often studied alongside Ly6C, as both are components recognized by the less specific Gr-1 antibody clone, RB6-8C5. For precise identification of mature neutrophils, the standard approach is to define them as cells that are high for both CD11b and Ly6G. This double-positive phenotype distinguishes them from most other cells in the myeloid compartment, allowing for a clear and consistent definition in experimental settings. Establishing this specific two-marker “gate” is the foundational step for any study seeking to quantify or characterize mouse neutrophils.
Tracking Neutrophil Maturation and Subsets
Neutrophil surface marker expression changes dynamically as the cells mature in the bone marrow and respond to inflammatory cues. This shifting pattern allows researchers to track the cell’s developmental stage and functional state, moving beyond simple identification. The relative expression of Ly6G and Ly6C is particularly informative for distinguishing between mature and immature cells. Mature circulating neutrophils are characterized by high Ly6G expression but low Ly6C expression.
Conversely, immature neutrophils or granulocytic progenitor cells in the bone marrow often display high expression of both Ly6G and Ly6C. This Ly6G-high/Ly6C-high phenotype is also observed in pathological states such as inflammation or cancer, where the rapid release of immature cells is termed “emergency myelopoiesis.” The presence of Ly6G-intermediate cells can further indicate a pool of resident or less mobile neutrophils, suggesting a spectrum of maturity and functional capacity in tissues like the spleen.
Markers of Operational Status
Other markers reflect the cell’s operational status, especially its migratory potential and age. Mature, circulating neutrophils express high levels of L-selectin (CD62L), which facilitates their initial tethering to the blood vessel wall before migration. As neutrophils age or become activated, they downregulate CD62L and upregulate the chemokine receptor CXCR4. CXCR4 is a marker associated with a return to the bone marrow for clearance. Analyzing a CD62L-low and CXCR4-high profile indicates an aged neutrophil population, providing deeper insight into the lifespan and clearance mechanisms of these cells.
Using Markers in Laboratory Techniques
The surface markers Ly6G and CD11b are the basis for powerful laboratory techniques used to quantify, isolate, and visualize neutrophils. Flow cytometry and Fluorescence-Activated Cell Sorting (FACS) are the most common methods, relying on fluorescently labeled antibodies specific to these markers. Researchers use these antibodies to stain a single-cell suspension, such as blood or bone marrow, and then run the cells through an instrument that measures the fluorescence intensity of each marker on every cell.
This process generates a scatter plot where Ly6G expression is plotted against CD11b expression, allowing researchers to visually identify and “gate” the double-positive neutrophil population. Using FACS, the instrument can physically separate the fluorescently marked cells, providing a highly purified population for downstream functional assays. The distinct physical properties of neutrophils, such as their high granularity, are also used in flow cytometry, as they exhibit high side scatter (SSC) characteristics.
For visualizing neutrophils directly within tissues, immunohistochemistry (IHC) and immunofluorescence (IF) techniques are employed. A tissue sample is sectioned, and antibodies against markers like Ly6G are used to locate the cells. The antibody is typically linked to an enzyme or a fluorescent dye, allowing the Ly6G-positive cells to be seen under a microscope. This visualization is invaluable for determining neutrophil location, density, and morphology within an inflamed organ, providing spatial context that complements numerical data.