Concentration of Rat Splenic CD4+CD25+ Regulatory T Cell by Magnetic Cell Sorting System in vitro_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

PU Liyong , WANG Xuehao , LI Xiangcheng , ZHANG Feng , YU Yue , YAO Aihua , CHENG Feng , LI Guoqiang.

Liver Transplantation Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China;

Corresponding author：

Keywords：

Magnetic cell sorting CD4+CD25+ regulatory T cell Flow cytometry technique Rat

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【Abstract】ObjectiveTo find a stable and efficient method for ex vivo concentration of CD4+CD25+ regulatory T cells in rats.
MethodsCD4+CD25+ regulatory T cells were separated from the rat splenic cells in two steps by magnetic cell sorting (MACS) system. CD4+ T cells were first negatively sorted by cocktail antibodies and antiIgG magnetic microbeads. And then CD4+CD 25+T cells were positively sorted by antiCD25 PE and antiPE magnetic microbeads. The purity and the cell survival rate of the sorted cells were measured by flow cytometry and trypan blue dyeing respectively, and the immunosuppressive action of CD4+CD25+ T cells on the proliferation of CD4+CD25- T cells was also assessed by in vitro cell proliferation assay.
ResultsThe purity of negatively sorted CD4+ T cells were (83.6±2.5)% (79%~87%), and the purity of positively sorted CD4+CD25+ T cells was (90.2±1.8)% (86%~93%) with the survival rate of (92.8±3.4)% (92%~95%). These concentrated cells significantly suppressed the proliferation of CD4+CD25- T cells in mixed lymphocyte culture （CD25+/CD25- versus CD25-, P＜0.01）.
ConclusionWe created a twostep procedure of magnetic cell sorting system for CD4+CD25+ regulatory T cells sorting, which insures the cells to be satisfactorily purified and well functioned.

Citation： PU Liyong,WANG Xuehao,LI Xiangcheng,ZHANG Feng,YU Yue,YAO Aihua,CHENG Feng,LI Guoqiang.. Concentration of Rat Splenic CD4+CD25+ Regulatory T Cell by Magnetic Cell Sorting System in vitro. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2006, 13(3): 275-278. doi: Copy

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2. [Ｊ]. J Immunol, 1995; 155(3)∶1151.
3. [Ｊ]. J Immunol, 1998; 160(3)∶1212.
4. [Ｊ]. Int Immunol, 1998; 10(12)∶1969.
5. [Ｊ]. Blood, 2002; 99(10)∶3493.
6. [Ｊ]. Blood, 2003; 102(6)∶2180.
7. [Ｊ]. J Exp Med, 2001; 193(11)∶1303.
8. [Ｊ]. Blood, 2004; 104(3)∶895.
9. Stephens LA, Barclay AN, Mason D. Phenotypic characterization of regulatory CD4+CD25+ T cells in rats.
10. Sakaguchi S, Sakaguchi N, Asano M, et al. Immunologic selftolerance maintained by activated T cells expressing IL2 receptor alphachains (CD25). Breakdown of a single mechanism of selftolerance causes various autoimmune diseases.
11. SuriPayer E, Amar AZ, Thornton AM, et al. CD4+CD25+ T cells inhibit both the induction and effector function of autoreactive T cells and represent a unique lineage of immunoregulatory cells.
12. Takahashi T, Kuniyasu Y, Toda M, et al. Immunologic selftolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state.
13. Taylor PA, Lees CJ, Blazar BR. The infusion of ex vivo activated and expanded CD4+CD25+ immune regulatory cells inhibits graftversushost disease lethality.
14. Jiang S, Camara N, Lombardi G, et al. Induction of allopeptidespecific human CD4+CD25+ regulatory T cells ex vivo.
15. Dieckmann D, Plottner H, Berchtold S, et al. Ex vivo isolation and characterization of CD4+CD25+ T cells with regulatory properties from human blood.
16. Hoffmann P, Eder R, KunzSchughart LA, et al. Largescale in vitro expansion of polyclonal human CD4+CD25+ high regulatory T cells.
17. Ikemoto T, Tashiro S, Yasutomo K, et al. Donorspecific tolerance induced by simultaneous allogeneic islet transplantation with CD4+CD25+ Tcells into hepatic parenchyma in mice [Ｊ]. J Med Invest, 2004; 51(3-4)∶178.
18. 徐勇, 霍梅. 免疫磁珠分离及流式细胞仪分选纯化外周血CD34+/CD90+ 干细胞 [Ｊ]. 临床检验杂志, 2004; 22(4)∶246.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Concentration of Rat Splenic CD4+CD25+ Regulatory T Cell by Magnetic Cell Sorting System in vitro

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