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Updated: Aug 23, 2025
Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells
Published on: November 28, 2015
Combinatorial Application of Papain and CD66B for Isolating Glioma- Associated Neutrophils
Xing Xu1,2, Yongchang Yang1,2, Yancheng Liu1,2
1Key Laboratory of Cancer Prevention and Therapy of Tianjin, Tianjin's Clinical Research Center for Cancer, Tianjin, China.
Background:
Stromal cells in the tumor microenvironment play crucial roles in glioma development. Current methods for isolating tumor-associated stromal cells (such as neutrophils) are inefficient due to the conflict between tissue dissociation and cell surface protein protection, which hampers the research on patient-derived stromal cells. Our study aims to establish a novel method for isolating glioma-associated neutrophils (GANs).
Methods:
To observe neutrophil-like polymorphonuclear cells, we performed Hematoxylin-Eosin staining on glioma tissues. For isolating single cells from glioma tissues, we evaluated the efficiency of tissue dissociation with FastPrep Grinder-mediated homogenization or proteases (trypsin or papain) digestion. To definite specific markers of GANs, fluorescence-activated cell sorting (FACS) and immunofluorescence staining were performed. FACS and Ficoll were performed for the separation of neutrophils from glioma tissue-derived single-cell or whole blood pool. To identify the isolated neutrophils, FACS and RT-PCR were carried out.
Results:
Neutrophil-like cells were abundant in high-grade glioma tissues. Among the three tissue dissociation methods, papain digestion produced a 5.1-fold and 1.7-fold more living cells from glioma mass than physical trituration and trypsin digestion, respectively, and it preserved over 97% of neutrophil surface protein markers. CD66B could be adopted as a unique neutrophil surface protein marker for FACS sorting in glioma. Glioma-derived CD66B+ cells specifically expressed neutrophil marker genes.
Conclusion:
A combination of papain-mediated tissue dissociation and CD66B-mediated FACS sorting is an effective novel method for the isolation of GANs from glioma tissues.
