Establishment and application of flow cytometry in detecting human cells in human/goat chimera models
10.3760/cma.j.issn.1009-9158.2009.04.021
- VernacularTitle:流式细胞术检测人/山羊嵌合体中人源细胞方法的建立与应用
- Author:
Juan WANG
;
Meijue CHEN
;
Zhijuan GONG
;
Zhaorui PEN
;
Fanyi ZENG
;
Shuzhen HUANG
- Publication Type:Journal Article
- Keywords:
Flow eytometry;
Hematopoietic stem cells;
Transplantation chimera;
Antigens,surface
- From:
Chinese Journal of Laboratory Medicine
2009;32(4):441-445
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish the methodology of flow cytometry for detecting human cells in human/goat chimerisra.Methods Human hemopoietic stem/progenitor cells (CD+34 cells) or MIG-tranadueed-GFP CD+34 cells were transplanted into the peritoneal cavity of fetal goats in utero to obtain human/goat chimera modeL The peripheral blood cells from the chimeras were labeled with multiple mouse anti-human antibodies and the monoelonal antibodies that were specific for human but had not or only minimal cross-reaction with goat were screened as the primary antibodies for routine analysis in flow cytometry.Human cord blood was proportionally (25% ,50% ,75%,100%) added into the blood of the untransplanted goats and the cells were labeled with CD+34 monoclonal antibody.The region and size of the "gate" were chosen based on to the distribution of CD+34 cells or human cord blood.One human/goat chimera marked with GFP (MIG goat) was sacrificed and the substantial liver cells from its perfused liver were analyzed for the GFP+cells percentage and DNA contents by flow cytometry.Results CD7,CD15,CD38,CD45CD20CD34CD14and GPA monoclonal antibodies were chosen as the primary antibodies in rou tine detection by flow cytometry.The size and area of the "gate" were also defined.29.1% (29100/100 000 ) of the substantial liver cells from the MIG goat expressed GFP.DNA content analysis showed that the GFP+ cells obtained from the liver of MIG goat mainly manifested two peaks that were correspond to those of human.Conclusions Flow cytometry is rapid,simple and effective for the investigation of differentiation,homing and biological characteristics of stem cells in vivo.The selections of suitable surface antibodies and the "gate" are very important for detecting human cells accurately in the human/goat chimerism.
