Objective: To assess the separation efficiency of magnetic-activated cell sorting in the purification of CD4+ T cells from murine spleen, and observe the effects of koumine on the proliferation of the separated cells.
Methods: CD4+ T cells were isolated from murine spleen by magnetic-activated cell sorting (MiniMACS). Fluorescence-activated cell sortering was employed to determine the purity of CD4+ T cells before and after the separation procedure followed by evaluation of the cell viability using trypan blue staining. Concanavalin A- (ConA, 5 microg/ml) or phytahematoagglutinin (PHA,1 mg/ml)-induced murine T cells were treated with different concentrations of koumine (10-320 microg/ml), and their proliferation was determined by MTT colorimetry, and enzyme-linked immunosorbent assay was used to measure IL-2 level in the cell culture supernatant.
Results: The purity of CD4+ T cells reached (90.3+/-5.8)% after the purification with a cell viability of (94.9+/-3.6)%. Koumine (20-320 microg/ml) dose-dependently inhibited ConA- or PHA-induced proliferation of murine lymphocytes as compared with the controls (P<0.05). Koumine (20, 100, and 200 microg/ml) significantly decreased the level of IL-2 in comparison with the control group (P<0.05).
Conclusions: CD4+ T cells of high purity can be obtained from murine spleen using MiniMACS without impairing the viability of the cells. Koumine significantly inhibits the proliferation of murine CD4+ T cells due to its immunosuppressive effect and inhibition of IL-2 secretion.
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