TY  - JOUR
TI  - Long-Term Neutrophil Depletion and Flow Cytometric Analysis of Brain Neutrophils in the 5xFAD Mouse Model
AU  - Huang, Haoning 
AU  - Bao, Xiaoping 
AU  - Deng, Qing 
VL  - 16
IS  - 21
PY  - 2026
DA  - 2026/11/05
SP  - e5864
C1  - Bio-protocol 2026;16:e5864
DO  - 10.21769/BioProtoc.5864
UR  - https://doi.org/10.21769/BioProtoc.5864
AB  - Neutrophils are the most abundant circulating leukocytes in human blood and have been implicated in the progression of Alzheimer’s disease (AD). Elevated neutrophil numbers have been observed in the blood and brain parenchyma of AD patients and mouse models, yet their precise pathological role remains poorly understood. A major barrier to progress is the lack of validated protocols for long-term neutrophil depletion and reliable recovery of brain-infiltrating neutrophils for characterization. Here, we describe a step-by-step protocol for sustained neutrophil depletion in the 5xFAD mouse model using intraperitoneal injection of an anti-Ly6G antibody for six weeks. We used a dose-escalation strategy to maintain depletion efficiency, as neutrophil regeneration increases over time. We also applied a previously established flow cytometry method to monitor neutrophil depletion in peripheral blood, using an antibody panel designed to avoid interference from the depleting anti-Ly6G clone. Furthermore, we present an optimized brain tissue processing workflow that combines mechanical dissociation with Percoll density gradient centrifugation to collect brain-infiltrating neutrophils for downstream flow cytometric analysis. Here, this workflow was applied and validated in the 5xFAD mouse model but is not specific to AD brain tissue and may be applicable to other mouse models.
KW  - 5xFAD
KW  - Neutrophil depletion
KW  - Neutrophil isolation
KW  - Flow cytometry
KW  - Anti-Ly6G
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False