Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells
Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells
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DOI：
10.3791/53676
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发表时间：
2015-11-01
影响因子：
1.2
通讯作者：
Lowenstein, Pedro R.
中科院分区：
文献类型：
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作者：
Baker, Gregory J.;Castro, Maria G.;Lowenstein, Pedro R.
我们的实验室最近证明，如果癌细胞缺乏 β-半乳糖苷结合凝集素 galectin-1 (gal-1) 的表达，自然杀伤 (NK) 细胞能够在颅内植入后很快根除原位植入的小鼠 GL26 和大鼠 CNS-1 恶性胶质瘤。最近的研究表明，Gr-1(+)/CD11b(+) 骨髓细胞群体对于这种效应至关重要。为了更好地了解 NK 和骨髓细胞合作产生 gal-1 缺陷型肿瘤排斥的机制，我们开发了一种用于分离和分析胶质瘤浸润外周血单核细胞 (PBMC) 的综合方案。该方法通过比较表达 gal-1 的 GL26 神经胶质瘤的肿瘤微环境中的 PBMC 浸润与通过 shRNA 敲低而导致 gal-1 缺陷的神经胶质瘤的肿瘤微环境的 PBMC 浸润进行了演示。该协议首先描述如何培养和准备 GL26 细胞以接种到同基因 C57BL/6J 小鼠大脑中。然后解释了从早期脑肿瘤微环境中分离和流式细胞术分析神经胶质瘤浸润 PBMC 所涉及的步骤。该方法适用于许多体内实验设计，其中需要有关免疫渗透到大脑中的时间数据。该方法灵敏且高度可重复，因为在肿瘤植入后 24 小时即可从颅内肿瘤中分离出胶质瘤浸润的 PBMC，并且在整个独立实验中从时间点匹配的肿瘤中观察到相似的细胞计数。单个实验人员可以在大约 4-6 小时内完成从脑采集到胶质瘤浸润 PBMC 的流式细胞术分析的方法，具体取决于要分析的样本数量。实验者也可以自行决定使用替代的神经胶质瘤模型和/或细胞特异性检测抗体来评估感兴趣的几种其他免疫细胞类型的浸润，而不需要改变整个程序。
Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the beta-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1(+)/CD11b(+) myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists' discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.