title: [Separation and amplification of CD4(+)CD25(+) regulatory T cells from sensitized mice].
abstract: The aim of this study was to separate and amplify CD4(+)CD25(+)Treg cells from splenocytes of sensitized mice. The percentage of CD4(+)CD25(+)Treg cells was detected by flow cytometry in sensitized and normal control mice. CD4(+)T, CD4(+)CD25(+)Treg and CD4(+)CD25(-) T cells were isolated from mouse splenocytes by MACS. CD4(+)CD25(+)Treg cells were expanded in vitro cultures in addition of CD3/CD28 MACSiBead and IL-2. The activity of cells was detected with 0.4 trypan blue staining. The purity of cells after sorting, the main surface marker and the level of Foxp3 were detected by flow cytometry. The results showed that CD4(+)CD25(+)Treg cell proportion was higher in sensitized mice than normal control mice (P < 0.05). The average purity of CD4(+)CD25(+)Treg cells was 87. The activity of these cells was more than 97, and the expression of Foxp3 in these cells was high. The amplification multiples achieved 42 times after 2 weeks in vitro. The percentage of CD4(+)CD25(+) regulatory T cells was 85.32, and the expression of Foxp3 decreased from (76.92 ± 1.72) to (75.33 ± 2.11) (P > 0.05). It is concluded that the sorting of CD4(+)CD25(+)Treg cells is isolated successfully by MACS without affecting the vitality of target cells. The amplification of CD4(+)CD25(+)Treg cells is successful in vitro. Expression of surface markers and Foxp3 gene does not obviously change after amplification, so that to establish a practical method to recover and enlarge the amount of CD4(+)CD25(+)Treg cells in good condition.
authors.affiliation: Department of Pediatrics, SUN Yat-Sen University, Guangzhou, China.
authors.forename: Li
authors.initials: L
authors.lastname: Pan
authors.forename: Wen-Jun
authors.initials: WJ
authors.lastname: Weng
authors.forename: Lü-Hong
authors.initials: LH
authors.lastname: Xu
authors.forename: Jing
authors.initials: J
authors.lastname: Wei
authors.forename: Jian-Pei
authors.initials: JP
authors.lastname: Fang
mesh_terms: D000818:Animals; D015704:CD4 Antigens / Q000096:biosynthesis; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors / Q000378:metabolism; D018189:Immunomagnetic Separation / Q000379:methods*; D053645:Interleukin-2 Receptor alpha Subunit / Q000378:metabolism; D018655:Lymphocyte Count; D051379:Mice; D008810:Mice, Inbred C57BL; D050378:T-Lymphocytes, Regulatory / Q000166:cytology*
chemical_list: D015704:CD4 Antigens; D051858:Forkhead Transcription Factors; C498833:Foxp3 protein, mouse; D053645:Interleukin-2 Receptor alpha Subunit