[Development of a CFSE-based flow cytometry for evaluating EIAV-stimulated proliferation of T lymphocytes].
OBJECTIVE
To develop a flow cytometry using (5-carboxyfluorescein diacetate succinmidyl ester, CFSE) to detect the proliferation of specific T lymphocytes from equine infectious anemia virus (EIAV).


METHODS
Peripheral blood mononuclear cells (PBMC) were isolated, stained with CFSE and incubated with EIAV for 5 days. After interacted with either CD4(+) or CD8(+) antibody, the cells were detected for proliferated population, which contained serially 2-fold reduced CFSE in CD4(+) and CD8(+) T lymphocytes.


RESULTS
The concentration of CFSE, and the type, concentration and reaction time of EIAV-specific antigens were tested and optimized to improve the sensitivity and specificity of this flow cytometry-based assay. PBMCs isolated from the horses infected with either virulent or vaccine strains of EIAV and health controls were analyzed to evaluate this technique. The proliferation rates of CD4(+) and CD8(+) T lymphocytes were observed in the cells from differently treated horses.


CONCLUSIONS
The CFSE-based flow cytometry is a reliable tool to analyze antigen-specific proliferation of lymphocytes, which plays an important role in evaluating the immune responses induced by EIAV and other viruses.
Lin
Yue-zhi
YZ
Division of Livestock Infectious Diseases, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150001, China.
Deng
Xi-lin
XL
Shen
Nan
N
Lü
Xiao-ling
XL
Zhao
Li-ping
LP
Kong
Xian-gang
XG
Shao
Yi-ming
YM
Zhou
Jian-hua
JH
D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000821:virology; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000821:virology; D049109:Cell Proliferation; D004859:Equine Infectious Anemia / Q000276:immunology / Q000821:virology; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry*; D006736:Horses; D004860:Infectious Anemia Virus, Equine / Q000502:physiology*; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology / Q000821:virology; D013388:Succinimides / Q000737:chemistry*; D013601:T-Lymphocytes / Q000276:immunology* / Q000821:virology
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D005452:Fluoresceins; D013388:Succinimides