Flow cytometric analysis of T lymphocyte proliferation in vivo by EdU incorporation.
Monitoring T lymphocyte proliferation, especially in vivo, is essential for the evaluation of adaptive immune reactions. Flow cytometry-based proliferation assays have advantages in measuring cell division of different T lymphocyte subsets at the same time by multicolor labelling. In this study, we aimed to establish the use of 5-Ethynyl-2'-deoxyuridine (EdU) incorporation in vivo to monitor T lymphocyte proliferation by flow cytometry with an adoptive transfer model. We found that fixation followed by permeabilization preserved T cell surface antigens and had no obvious effects on the fluorescence intensity of APC, PE, PE-Cy7, FITC and PerCP-Cy5.5 when the concentration of the permeabilization reagents was optimized. However, the click reaction resulted in a significant decrease in the fluorescence intensity of PE and PE-Cy7, and surface staining after the click reaction improved the fluorescence intensity. Thus, an extra step of blocking with PBS with 3% FBS between the click reaction and cell surface staining is needed. Furthermore, the percentage of EdU-positive cells increased in a dose-dependent manner, and the saturated dose of EdU was 20mg/kg. Intraperitoneal and intravenous injection had no differences in lymphocyte proliferation detection with EdU in vivo. In addition, T cell proliferation measured by EdU incorporation was comparable to BrdU but was lower than CFSE labelling. In conclusion, we optimized the protocols for EdU administration in vivo and staining in vitro, providing a feasible method for the measurement of T lymphocyte proliferation with EdU incorporation by flow cytometry in vivo.
Experimental and Translational Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China; Beijing Key Laboratory of Tolerance Induction and Organ Protection in Transplantation, Beijing 100050, China; National Clinical Research Center for Digestive Diseases, Beijing 100050, China.
Xiaojing
X
Sun
Experimental and Translational Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China; Beijing Key Laboratory of Tolerance Induction and Organ Protection in Transplantation, Beijing 100050, China; National Clinical Research Center for Digestive Diseases, Beijing 100050, China.
Chunpan
C
Zhang
Experimental and Translational Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China; Beijing Key Laboratory of Tolerance Induction and Organ Protection in Transplantation, Beijing 100050, China; National Clinical Research Center for Digestive Diseases, Beijing 100050, China.
Hua
H
Jin
Experimental and Translational Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China; Beijing Key Laboratory of Tolerance Induction and Organ Protection in Transplantation, Beijing 100050, China; National Clinical Research Center for Digestive Diseases, Beijing 100050, China.
Guangyong
G
Sun
Experimental and Translational Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China; Beijing Key Laboratory of Tolerance Induction and Organ Protection in Transplantation, Beijing 100050, China; National Clinical Research Center for Digestive Diseases, Beijing 100050, China.
Yue
Y
Tian
Experimental and Translational Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China; Beijing Key Laboratory of Tolerance Induction and Organ Protection in Transplantation, Beijing 100050, China; National Clinical Research Center for Digestive Diseases, Beijing 100050, China.
Wen
W
Shi
Experimental and Translational Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China; Beijing Key Laboratory of Tolerance Induction and Organ Protection in Transplantation, Beijing 100050, China; National Clinical Research Center for Digestive Diseases, Beijing 100050, China. Electronic address: zhangd2010@hotmail.com.
Dong
D
Zhang
D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D057930:Click Chemistry; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000494:pharmacology; D008297:Male; D008810:Mice, Inbred C57BL; D017830:Octoxynol / Q000494:pharmacology; D012503:Saponins / Q000494:pharmacology; D013194:Staining and Labeling / Q000379:methods*; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism
D005456:Fluorescent Dyes; D012503:Saponins; D017830:Octoxynol; C031086:5-ethynyl-2'-deoxyuridine; D003857:Deoxyuridine
BrdU; CFSE; Cell proliferation; EdU; Flow cytometry