title: Multicolor flow cytometry analysis of the proliferations of T-lymphocyte subsets in vitro by EdU incorporation.
abstract: EdU (5-ethynyl-2'-deoxyuridine) incorporation has proved advantageous in the studies of cell kinetics, DNA synthesis, and cellular proliferation in vitro and in vivo compared to [(3) H]thymidine incorporation and BrdU (5-bromo-2'-deoxyuridine) incorporation. Here, we describe a method that combines EdU incorporation and immunostaining with flow cytometric analysis to detect the proliferations of T lymphocyte subsets in vitro and optimized the assay's conditions. We found that the number of EdU(+) cells were associated with EdU concentration, incubation time, and the volume of Click reaction solution, the best EdU concentration 10-50 μM, the optimal incubation time 8-12 h and the proper volume of Click volume 100 μl for labeling 1 × 10(6) lymphocytes. Fixation was better to be performed before permeabilization, not together with. Furthermore, the permeabilization detergent reagent, PBS with 0.05% saponin was better than Tris buffer saline (TBS) with 0.1% Triton X-100. In addition, sufficient wash with PBS with 0.05% saponin has no influence on the staining of EdU(+) cells. Also, the lymphocytes incorporating EdU could be stored at 4°C, -80°C, and in liquid nitrogen up to 21 days. The present study will aid in optimization of flow cytometry assay to detect the proliferations of T cell subsets by EdU incorporation and the labeling of cell surface antigens.
authors.affiliation: Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, and National Center for Clinical Laboratories, Beijing Hospital of the Ministry of Health, Beijing, People's Republic of China.
authors.forename: Yanli
authors.initials: Y
authors.lastname: Sun
authors.forename: Yu
authors.initials: Y
authors.lastname: Sun
authors.forename: Guigao
authors.initials: G
authors.lastname: Lin
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.forename: Kuo
authors.initials: K
authors.lastname: Zhang
authors.forename: Jiehong
authors.initials: J
authors.lastname: Xie
authors.forename: Lunan
authors.initials: L
authors.lastname: Wang
authors.forename: Jinming
authors.initials: J
authors.lastname: Li
mesh_terms: D000818:Animals; D001692:Biological Transport; D001973:Bromodeoxyuridine / Q000737:chemistry / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D057930:Click Chemistry; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D005404:Fixatives; D005434:Flow Cytometry / Q000379:methods*; D018655:Lymphocyte Count; D051379:Mice; D008810:Mice, Inbred C57BL; D013154:Spleen / Q000166:cytology; D013194:Staining and Labeling / Q000379:methods*; D016176:T-Lymphocyte Subsets / Q000737:chemistry* / Q000166:cytology*; D013936:Thymidine / Q000737:chemistry / Q000378:metabolism
chemical_list: D005404:Fixatives; D001973:Bromodeoxyuridine; C031086:5-ethynyl-2'-deoxyuridine; D013936:Thymidine; D003857:Deoxyuridine