Establishment of 14-Color Panel for Determining Leukocyte Subsets in Human Peripheral Blood with Flow Cytometry

• Published in: Sichuan da xue xue bao. Journal of Sichuan University. Yi xue ban Vol. 53; no. 1; pp. 127 - 132
• Main Authors: Huang, Qiao-Rong, Jiang, Yan-Ni, Li, Hui-Fang, Li, Xue, Meng, Wen-Tong
• Format: Journal Article
• Language: Chinese
• Published: China 01.01.2022
• Subjects: Cell Count; Flow Cytometry; Humans; Immunophenotyping; Leukocytes; Lymphocyte Subsets; Multicolor flow cytometry; Immunophenotype; Cell subset; Panel optimization; Human peripheral blood
• ISSN: 1672-173X
• DOI: 10.12182/20220160106

To establish a 14-color flow cytometry protocol for the examination of leukocyte subsets in human peripheral blood. We used cell membrane surface antibodies CD45, CD3, CD4, CD8, CD19, CD56, CD16, CD14, CD25, CD127, HLA-DR, CD123, CD11c and nucleus staining dye DAPI to establish a 14-color flow cytometry assay to determine the major cell subsets in human peripheral blood. We collected peripheral blood specimens from healthy volunteers to test for antibody titers and optimal photomultiplier tube (PMT) voltage, and to conduct single-color staining and fluorescence minus one control staining. After determining the test method and test conditions, the peripheral blood samples of 18 healthy volunteers were analyzed. According to the cell classification and staining index, optimal antibody mass concentrations selected were as follows: CD25 and CD127 at 8.0 μg/mL, CD45, CD3, CD14 and CD123 at 4.0 μg/mL, CD8, CD19, CD56, CD16, HLA-DR and CD11c at 2.0 μg/mL, CD4 at 1.0 μg/mL and DAPI at 0.1 μg/mL. The detection voltage