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Aspirate all the contents into a 1 mL syringe, remove the needle, replace with the syringe filter, and plunge the contents into the digestion buffer. In order to do this, add 900 μL digestion buffer into the Eppendorf containing 100 μL of collagenase at 20,000 units/mL in DW. Collagenase will need to be passed through a 0.45 μm syringe filter once dissolved in DW. To prepare the digestion buffer, add 40 μL of 50 mg/mL of DNase stock dissolved in DW into 15 mL of TCM. T Cell Medium (TCM): 500 mL RPMI 1640, 50 mL FBS, 5.5 mL Pen/Strep, 5.5 mL NEAA, 5.5 mL Na-pyruvate, 5.5 mL glutamine, 550 μL β-mercaptoethanol, 550 μL gentamycin.ĭigestion Buffer composition (per bag per tumor): 15 mL TCM, 40 μL of 50 mg/mL stock of DNase I grade II from bovine pancreas (Roche Cat#10104159001), 100 μL of 20,000 unit/mL stock Collagenase Type IV powder (Gibco-Life Technologies Cat#17104–019), both prepared in distilled water (DW). Additionally, we provide insight regarding acquisition of the samples and gating strategies on a cytometer (Subheading 3.7). Although more selective methods may be appropriate if gene expression analysis, proteomic profiling, or other investigations requiring a homogeneous end product are pursued, this protocol works exceedingly well for flow cytometric analyses of TILs.īriefly, this method entails preparing the materials needed (Subheading 3.1), harvesting the brain tumor (Subheading 3.2), mechanically and enzymatically digesting the tumor (Subheading 3.3), lysing red blood cells (RBCs) and preparing the TILs (Subheading 3.4), stimulating the TILs in the presence of phorbol 12-myristate 13-acetate (PMA)/ionomycin if intracellular cytokine staining is desired (Subheading 3.5), as well as washing and preparing the cells for staining (Subheading 3.6). We find that staining for surface and intracellular markers on T cells by flow cytometry is not affected by the presence of either tumor or brain cells. For this reason, we have omitted these selective steps in our procedure. This loss is of particular importance in the setting of brain tumors due to lack of overwhelming T cell infiltration.
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Other TIL isolation procedures include use of Ficoll gradient, magnetic beads, or FACS sorting and result in significant loss of the population of interest. There are many procedures for TIL isolation described in the literature for a variety of solid tumors, though we have found that the following protocol is best suited to brain tumors, including glioblastoma (GBM). This chapter describes a relatively rapid isolation of tumor-infiltrating lymphocytes (TILs) from brain tumors, as well as procedures for functional profiling and flow cytometry following TIL isolation.