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. 2021 Dec:499:113158.
doi: 10.1016/j.jim.2021.113158. Epub 2021 Sep 29.

Broad immunophenotyping of the murine brain tumor microenvironment

W H Tomaszewski  1 , J Waibl-Polania  2 , A M Miggelbrink  2 , M A Chakraborty  3 , P E Fecci  4 , J H Sampson  4 , M D Gunn  5
Affiliations

Broad immunophenotyping of the murine brain tumor microenvironment

W H Tomaszewski et al. J Immunol Methods. 2021 Dec.

Abstract

Here we present a 14-color flow cytometry panel for the evaluation of 13 myeloid and lymphoid populations within murine glioblastoma samples. Reagents, processing protocols, and downstream analyses were thoroughly validated and optimized to resolve the following populations: T cells (CD4, CD8, CD3), B cells (B220), NK cells (NK1.1), neutrophils (Ly6G), classical and non-classical monocytes (Ly6c, CD43), macrophages (F4/80, CD11b), microglia (CD45-lo, CD11b), and dendritic cells (DCs) (CD11c, MHC class II). In addition, this panel leaves Alexa Fluor 488/FITC open for the inclusion of fluorescent reporters or congenic marker staining.

Keywords: Glioblastoma; Multicolor flow cytometry; Preclinical; Tumor microenvironment; Unbiased cell classification.

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Figures

Figure 1 -
Figure 1 -
Graphical summary of unbiased cell type identification workflow
Figure 2 -
Figure 2 -
Representative gating strategy and tumor immune infiltrate analysis. (A) Singlets are defined, followed by leukocytes, and then lymphocytes and myeloid cells are separated. (B) Lymphocyte gating strategy. (C) Myeloid gating strategy. (D) Summary of tumor immune infiltrate from n = 5 mice. Results are representative of 2 experimental repeats.
Figure 3 -
Figure 3 -
Representative UMAP clustering results and unbiased tumor immune infiltrate analysis. (A) UMAP projection of CD45+ Live/Dead− cells from n=5 mice. (B) Heatmap of antigen targets included in the panel to guide cluster annotation. (C) Lineage markers projected into UMAP space. (D) Summary of tumor immune infiltrate from n = 5 mice. Results are representative of 2 experimental repeats.
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