Flow Cytometry gating for DN T cells. Cells isolated from murine lungs were cultured in 5% fetal bovine serum in RPMI
medium with 100 mg of Collagenase D for 30 minutes. Cells were strained and lymphocytes were isolated with percoll extraction.
Lymphocytes were stained with CD45 (APC-Cy7), TCR αβ (BV421), CD4+ (APC), CD8+ (FITC) antibodies and analyzed
through LSR-II flow cytometry. The lymphocyte population was isolated from SSC-A and FSC-A. CD45+ TCR+ subpopulation was selected
to visualize CD4+, CD8+, Double-Positive (CD4+CD8+) and Double-Negative (CD4−CD8−) T cells. Double-Negative T cells
are represented as the population within the red circle.