Establishment of a Novel Multi-Color Panel for Analyzing Treg Cell Subsets in Peripheral Blood of Patients with Dermatomyositis
HUANG Yingying1, JIANG Yu2, XING Yueting1, LI Yanwei1, GUO Chun1, WANG Jiajia1, XU Qin3*
To establish a new panel for Treg subsets in peripheral blood of patients with dermatomyositis, a 15-color multiparameter flow cytometric panel aimed to construct and optimize. Peripheral blood of dermatomyositis patients and healthy donors were collected and PBMCs were isolated by density gradient centrifugation. The antibody concentration, staining time, staining temperature, effect of light on antibody staining index, fluorescein exposure time and voltage were optimized for antibody staining and acquisition of PBMC cells on flow cytometry. Finally, a high dimensional panel with 15 colors were successfully established and targeted Treg subsets were analyzed with advanced application in FlowJo. The results showed that (1) the optimal voltage value of the panel can be established by blank cross method combined with staining index method; (2) the optimal antibody concentration of TIGIT BV421, HLA-DR PE, CD127 BV605, CD103 R718, CD25 PE-Cy7 after titration were 1.25, 1.25, 3, 2.5, 5 μL/test (100 μL staining volumn/test) respectively; (3) with this multicolor flow cytometry panel, antibody staining should be incubated at 4 °C for 30 min in dark; (4) and the samples should be aquired within 24 h. The established 15-color flow cytometry panel can successfully analyze the portion of Treg cell subsets and activation related molecules in human PBMC samples. This method is feasible for the analysis of peripheral Treg subsets in patients with in dermatomyositis and other disorders featured with treg deficiency.
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