Neutrophil Flow Cytometry Markers

Abstract: Neutrophils are primary protectors against pathogens. The amount and function are very important for infection prevention. Stimulation of microorganisms produces reactive oxygen species(ROS), releasing neutrophil elastase(NE) and myeloperoxidase(MPO). Cleavage of histones by NE promotes chromatin condensation. Binding between MPO and chromatin enhances unfolding of structure. Finally, chromatin and anti-microbial proteins make up of neutrophil extracellular traps(NETs). This process is called NETosis. NETs can capture and kill bacteria, fungi, parasite and viruses. Anti-microbial effects are very important. Currently, identification for neutrophil flow cytometry markers focuses on phagocytosis and oxidative burst.

Keywords: NETosis Assay, Neutrophil Flow Cytometry Markers, Infection Prevention, Neutrophils Test

1. New Evaluation Methods

Timmer KD et al. proposed new methods for evaluating phagocytosis, ROS production, ectodomain shedding and degranulation in Cells. CD62L shedding reflects activation status. Molecules like CD16b regulate immune response. Degranulation releases anti-microbial components(e.g. MPO, lysozyme) through CD66b labeled secondary particles. This method can synchronously detect multiple functions of neutrophil flow cytometry markers.

2. NETosis Assay

2.1. Flow Cytometry for NETosis in Bone Marrow(BM)

Detect PMNs in bone marrow with flow cytometry. 30nM PMA stimulated - NETosis for 4h. Set control group without 4h processing. First, set gating via FSC-A/FSC-H and SSC-A/SSC-H to remove cell doublets. Select PMN cell population of SSChiLy6G+. Finally, analyze SO+DAPI+ PMN cell population in Ly6G+ cell further, recognizing NETosis in neutrophils.

2.2. Flow Cytometry for NETosis in Purified PMNs

Stimulate with different concentrations of PMA. Stain with anti-CD66, DAPI and SO. Then, analyze NETosis with flow cytometry. First, in Figure 3A, gating is used to remove cell doublets. Then, in Figure 3B, gating via FSC/SSC(R1) eliminate contamination of lymphocytes and monocytes. In Figure 3C, identify PMNs according to SSChi feature and expression of CD66. Finally, Figure 3C and 3D show the proportion of SO+DAPI+ PMN in treated PMA group obviously increases. The background signal of untreated group is about 7%-20%.

3. General Phenotypic Analysis of NETs in Human Peripheral Cells

3.1. Gating for Quantitative Stimulation of NET-attached Neutrophils in Vitro

Gating via SSC-AhiFSC-Ahi removes cell doublets, and then identifies neutrophils via double positive CD15 and CD66b. Active neutrophils-attached DNA structure is recognized as CD15+CD66b+Zombie NIRdim7-AAD+. NET+ cell is MPO+NE+. The overall phenotype is CD15+CD66b+7-AAD+Zombie NIRdimMPO+NE+. DNase I can validate specificity of NETs. Besides, co-expression of H3cit and MPO/NE is the sign of PAD4-dependent NETosis.

3.2. In Vitro Monitoring of NETosis

Centrifuge and separate granulocyte in whole blood via density. NETotic neutrophils in 7-AAD+Zombie NIRdimMPO+NE+ show dosage-dependent increase after 2h stimulation by 0-7.1nM PMA(Figure 5A). Necrotic cells are 7-AAD+Zombie NIR+MPO++NE++, illustrated by red arrow. Annexin V detection shows NETotic cells don’t express apoptotic markers(Figure 5B). After stimulation by FSL-1, stain with seven-color NET Panel(Figure 5C) and CD11b/CD66b(Figure 5D) respectively to validate NETosis and activation state. Figure 5E shows different definitions of NETs affect 5.0nM PMA.

The common detection of fluorescence microscope for NETs is subjective and inefficient. High-throughput and reproducible flow cytometry for NETosis detection can improve analytical efficiency and accuracy.

REFERENCES

[1]CD177+ neutrophils drive extracellular matrix remodelling and HGF-alpha release in ALPPS-induced liver regeneration, PMID: 41390175.
[2]Microbiota-shaped neutrophil senescence regulates sexual dimorphism in bladder cancer, PMID: 40217111.