ABSTRACT
Objective • To optimize the method of the flow cytometry MoFlo Astrios EQ on single-cell sorting in 96-well plate. Methods • Using different aperture nozzles and sorting ways, the 32D, U937, iBMDM and 293T cells were used for single-cell sorting after the precise adjustment of the instrument and various parameters. The hole numbers with single cell and single-cell clones were detected after sorting. Results • In the single-cell sorting mode, the hole numbers with single cell were 83-91 by 70 μm nozzle and 87-93 by 100 μm nozzle. After 7-10 days of culture, the hole numbers with single-cell clones were 36-58 by 70 μm nozzle. In 100 μm nozzle, the hole numbers with single-cell clones were 53-78 by electrostatic charge sorting and 69-81 by straight-down sorting, respectively. Conclusion • In single-cell sorting, a better cell viability and higher cloning rate are observed in 100 μm nozzle and straight-down sorting.
ABSTRACT
Objective To isolate HIV-specific T cell clone and to expand them in vitro through the activation-induced expression of CD137 molecule.Methods The peripheral blood mononuclear cells were isolated from HIV-infected patients and HIV Gag specific CD3+CD8+CD137+T cell subset were sorted to 96-well plate in 1 cell/well by multicolor flowcytometry and single cell sorting.After 14 days in vitro culture with feeder cells and cytokines, the numbers and phenotypes of the cultured HIV-specific T cells were calcu-lated and identified.Results The CD137 expression was low on rested T cells but up regulated by the stim-ulation with Gag peptide pool.The CD8+CD137+T cells could secret IFN-γ.The number of CD8 T cells reached to 106 after 14 days in culture and expanded to 107-108 cells after 28 days of culture in vitro 100%of the cells remained activated upon Gag stimulation.Conclusion In stead of using IFN-γ, CD137 could be utilized as a novel molecule to isolate and expand HIV specific T cells in vitro.The expanded antigen spe-cific T cell clones could maintain good activation status.