Optimization and characterization of a protocol for sorting mouse tumor myeloid-derived suppressor cells by flow cytometry
10.19405/j. cnki. issn1000–1492.2026.07.001
- VernacularTitle:流式细胞术分选小鼠肿瘤髓源性抑制细胞的方案优化及鉴定
- Author:
Jia DU
1
;
Lulu RAO
1
;
Xin HOU
2
;
Yang MA
1
Author Information
1. School of Pharmacy， Anhui Medical University， Key Laboratory of Anti-inflammatory and Immune Medicine of the Ministry of Education， Anhui Collaborative Innovation Center of Anti-inflammatory and Immune Medicines， Rheumatoid Arthritis Research Center of Anhui Medical University， Hefei 230032
2. Department of Pathogen Biology and Immunology， School of Medicine， Ningbo University， Ningbo 315211
- Publication Type:Journal Article
- Keywords:
myeloid-derived suppressor cells;
flow cytometry;
cell sorting;
protocol optimization;
Lewis lung carcinoma;
tumor microenvironment
- From:
Acta Universitatis Medicinalis Anhui
2026;61(7):1163-1169
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo optimize the flow cytometric sorting workflow for myeloid-derived suppressor cells （MDSCs） from murine Lewis lung carcinoma （LLC） subcutaneous tumor tissues， improving single-cell suspension preparation efficiency and sorting purity， thereby providing high-quality cells for subsequent functional studies. MethodsSubcutaneous LLC tumor tissues were harvested from C57BL/6 mice， and two enzymatic digestion protocols were first compared to evaluate their effects on single-cell suspension preparation. After staining with a viability dye （Live/Dead） and fluorochrome-conjugated anti-mouse antibodies against CD45， CD11b， and Gr-1， cells were loaded onto a Beckman CytoFLEX cell sorter. Next， conventional and optimized gating strategies were applied. In the optimized strategy， the CD45+ population was used as the initial gate to directly define an MDSC-enriched region， followed by selection of CD11b+Gr-1+ cells. Finally， purity was assessed by flow cytometry， viability was determined by trypan blue staining， and RT-qPCR was performed to measure the expression of MDSC signature genes， including Arg1， Nos2， IL-10， and S100A8/A9. ResultsThe optimized enzymatic digestion protocol significantly increased the yield and viability of single cells. After optimization of the gating strategy， the post-sort positivity rate （purity） was markedly improved. Arg1， Nos2， IL-10 and S100A8/A9 were highly expressed in the sorted cells， indicating that the isolated cells retained characteristic MDSC features and functional signatures. ConclusionBy optimizing both the tissue digestion protocol and the flow cytometric gating strategy， we establish an efficient workflow for isolating MDSCs from tumor tissues. This method improves the yield and purity of single-cell preparations while preserving antigen integrity， providing a reliable methodological foundation for subsequent studies on MDSC function and metabolism.
