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Updated: Jul 13, 2026
Mouse Naïve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets
Published on: April 16, 2015
[Biological characteristics of human peripheric CD4+ CD25- T cells isolated and cultured in vitro]
Miao-shang Su1, Man-huan Xu, Fu-jiang Chen
1Department of Pediatrics, Second Affiliated Hospital of Wenzhou Medical College, Wenzhou 325027, China. sumish@163.com
Aim:
To isolate peripheric CD4(+) CD25(-) T cells in vitro, and study their biological characteristics.
Methods:
Human peripheric blood CD4(+) CD25(-) T cells were isolated by discontinuous density gradient centrfugal and dynal immunomagnetic beads, and then divided into three groups: control group(A), LPS group (B) and LPS+TGF-beta1 mAb group (D), After they were prepared 4 h, 3 d and 5 d, the percentage of CD4(+) CD25(+) T cells were tested by FCM, the levels of TGF-beta1 in cultured supernatants were detected by ELISA, and the expression of FOXP3 mRNA was examined by RT-PCR.
Results:
(1)LM showed CD4(+) CD25(-) T cells were of spherical cell nucleus, After CD4(+) CD25(-) T cells were cultured by anti-CD3/CD28 in vitro, their cellular volume increased and cytoplasm contained more particles. TEM showed CD4(+) CD25(-) T cells were of oval or kidney-shaped caryon. (2)FCM showed the purity of CD4(+) CD25(-) T cells was 91.5%-96%. The trypan blue experiment showed the energometry before and after detachment had no obvious difference(P>0.05). (3)FCM showed the percentage of CD4(+) CD25(+) T cells in B5d group(55.99+/-1.42)% increased markedly compared with that in A5d group(1.29+/-0.04)%. (4)ELISA showed the levels of TGF-beta1 in B3d(1.60+/-0.09) microg/L, in B5d(1.83+/-0.14) microg/L were significantly increased compared with those in A3d (0.35+/-0.04) microg/L and A5d(0.33+/-0.08) microg/L(P<0.01); but the levels in group D and group A had no significant difference; (5)RT-PCR showed FOXP3 mRNA in B3d group(0.84+/-0.07) and B5d group(1.85+/-0.24) increased strikingly compared with those in A3d(0.05+/-0.02) and A5d(0.04+/-0.02)(P<0.01), but those in group A and group D had no significant difference. (6)There was significantly positive correlation between the expression of TGF-beta1 and FOXP3 mRNA in the induced CD4(+) CD25(-) T cells (r=0.812, P<0.01).
Conclusion:
Human peripheric blood CD4(+) CD25(-) T cells can be isolated in vitro by discontinuous density gradient centrfugal and dynal immunomagnetic beads. The expression of FOXP3 mRNA in E.coli LPS-induced human CD4(+) CD25(-) T cells may correlate with the expression of TGF-beta1.
