Tumor-Infiltrating Lymphocyte Analysis by Flow Cytometry

The infiltration of lymphocytes in tumor tissue was analyzed by flow cytometry, as described elsewhere (20 (link)). To generate single cell from the tumor tissue (100-200 mg), Collagenase IV (Yeasen Biotech, Shanghai, China) was employed to digest the tissue in HBSS at 37°C for 30 minutes. The single cells were obtained by filtering the mixture through a 70 μm cell strainer (BD, Franklin Lakes, USA). Then, surface staining with anti-mouse antibodies for CD45 (GK1.5), CD4 (30-F11) and CD8 (53-6.7) was carried out on the single cells in the dark at 4°C for 30 min. After washing twice with PBS, the stained cells were fixed in a Foxp3/Transcription Factor Staining Buffer Set (eBioscience, San Diego, USA) in the dark at 4°C for 30 min. Subsequently, the fixed cells were permeabilized and stained with antibodies for IFN γ (XMG1.2) in the dark at 4°C for 30 min. Finally, the cells were washed twice, suspended in fluorescence-activated cell sorting (FACS) buffer, and analyzed using a FACS Calibur flow cytometer (BD, Franklin Lakes, USA). FlowJo software (TreeStar, Ashland, USA) was utilized to interpret the results.