In vitro EdU labeling of peripheral blood mononuclear cells in rabbits
10.3969/j.issn.2095-4344.2017.09.022
- VernacularTitle:EdU标记兔外周血单个核细胞的体外增殖
- Author:
Minglei ZHAO
;
Dongqin ZHEN
;
Jianfa HUANG
;
Weihua LI
;
Wencong WANG
;
Zhiquan LI
;
Hening ZHANG
;
Bikun XIAN
;
Yuting PENG
;
Minyi ZHOU
;
Bing HUANG
- From:
Chinese Journal of Tissue Engineering Research
2017;21(9):1432-1438
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: The proliferation of peripheral blood stem cells among peripheral blood mononuclear cells (PBMCs) invitro remains unclear. There is no optimal marker for tracing PBMCs transplanted in vivo.OBJECTIVE: To observe the degree of PBMC proliferation in stem cell medium by EdU labeling and to explore thefeasibility of EdU-labeled peripheral blood stem cells.METHODS: New Zealand rabbit PBMCs were isolated and cultured for 1 to 5 days in stem cell medium supplementedwith EdU. The cells were observed and counted at 0, 1, 2, 3, 4 and 5 days in culture. The cells were harvested at eachtime point and stained with EdU fluorescent reagents. Then, confocal microscopy and flow cytometry were used to detectEdU-labeled cells.RESULTS AND CONCLUSION: (1) Freshly isolated rabbit PBMCs were rounded and showed clear outline. After 1 dayculture, most of the cells were suspended in the medium, spherical or round. There were also a few cell clusters andadherent cells scattered in a triangle or polygon shape; after 2 days culture, more cell debris were observed, and mostcells were round; when cultured for 3-5 days, increased cell debris, smaller cell mass and decreased cell densitysignificantly were observed. (2) With the prolongation of culture time, the cell count decreased gradually. (3) Whencultured for 1 day, EdU labeled cells in red were scattered. The number of cells marked with EdU red label increasedsignificantly at day 2 and remained unchanged after 3 days of culture. At 5 days of culture, the number of red cellsmarkedly decreased; the highest positive rate of EdU-labeled cells was (2.38±0.10)% at 2 days after culture. To conclude,these results showed that the proportion of proliferating cells in rabbit PBMCs was very low. EdU is capable of labelingproliferative cells among PBMCs.
