Therefore, a reliable 21-color flow cytometry protocol was established to detect the immune cell subsets in human non-small cell lung cancer (NSCLC) tumor tissues.<h4>Methods</h4>Cell membrane surface antibodies cluster of differentiation (CD)45, CD3, CD19, CD4, CD8, programmed cell death 1 (PD-1), CD39, CD103, CD25, CD127, chemokine receptor 8 (CCR8), CD56, CD11c, human leukocyte antigen (HLA)-DR, CD38, CD27, CD69, CD62L, CD45RA, CCR7 and nucleic acid dye L/D were used to develop the protocol. The gene discussed is PDCD1; the disease is non-small cell lung carcinoma.