@article {364301,
	title = {Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells},
	journal = {J Vis Exp.},
	volume = {28},
	number = {105},
	year = {2015},
	abstract = {Our laboratory has recently demonstrated that natural killer (NK)\&nbsp;cells\&nbsp;are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer\&nbsp;cells\&nbsp;are rendered deficient in their expression of the β-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+\&nbsp;myeloid\&nbsp;cells\&nbsp;is critical to this effect. To better understand the mechanisms by which NK and myeloid\&nbsp;cells\&nbsp;cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the\&nbsp;isolation\&nbsp;and\&nbsp;analysis\&nbsp;of\&nbsp;glioma-infiltrating\&nbsp;peripheral\&nbsp;blood\&nbsp;mononuclear\&nbsp;cells\&nbsp;(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\&nbsp;how to culture and prepare GL26\&nbsp;cells\&nbsp;for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the\&nbsp;isolation\&nbsp;and\&nbsp;flow\&nbsp;cytometric\&nbsp;analysis\&nbsp;of\&nbsp;glioma-infiltrating\&nbsp;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\&nbsp;glioma-infiltrating\&nbsp;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\&nbsp;flow\&nbsp;cytometric\&nbsp;analysis\&nbsp;of\&nbsp;glioma-infiltrating\&nbsp;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{\textquoteright} discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.},
	author = {GJ Baker}
}
