| Summary: | We presented a flow cytometry protocol to isolate and measure the cellular component of tumour immune microenvironment (TIME) of mice bearing breast cancer models. The immune cells infiltrating the tumour microenvironment (TME) have been getting more attention as the determination of their pro-or/and anti-tumorigenic activity contributes to cancer treatment success. In addition, our group has a strong interest to investigate the infiltration of eosinophils within the TME as their tumorigenic role is still unclear. This study aims to measure the immune cells infiltrating the TME, including eosinophils using flow cytometry with 6-colour detection. The single-cell suspensions derived from tumour sections of mice bearing EMT6 tumour model were harvested (n=4) and treated with CD45, Siglec-F, I-A/I-E, CD 11b, and Ly6G antibodies. A gating protocol was used based on the reported publications. Results showed that immune cells in the tumour section were detected by positive staining to CD45. Neutrophils were isolated based on Ly6G+, while the remaining granulocytes, such as macrophage and dendritic cells (DC), were isolated based on I-A/I-E+SCChi, and eosinophils on the positive expression of Siglec F. Overall, our tumour model presented with the highest percentage of neutrophils (63.05 ± 5.61%), while eosinophils constitute 1.64 ± 0.75% of the total population. In conclusion, our flow cytometric protocol with 6 colour detection are able to isolate and measure several immune cells within the TME, including eosinophils from the tumour section. © 2022, University of Malaya. All rights reserved.
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