Aim: To measure the in vitro cell division ability of CD8(+);NKT cells by CFSE staining and flow cytometry(FCM).
Methods: Fresh spleen lymphocytes of C57BL/J mice were separated and stained with CFSE, and then stimulated by ConA and LPS for 3 d, and by SEB for 5 d and 10 d respectively. The stimulated cells were harvested and analyzed for CD69 expression on the cell surface and the ability of cell division using FCM. The SEB-activated effector cells for 10 d further stimulated with IL-2 for the consecutive 10 days, and were analyzed for their cell division ability, CD69 expression and NKT cell subsets by FCM.
Results: ConA, LPS and SEB stimulated the proliferation of spleen cells. ConA and LPS made the cells divide 3 times within 3 d, and increased CD69 expression up to 74.19% and 41.56% respectively. SEB made the cells divide 5 times within 5 d and 7 times within 10 d respectively, with increased CD69 expression of 32.09% and 48.66% respectively. Ten-day IL-2 stimulation of SEB-activated cells caused population expansion for 7 times with the CD8(+);NKT cell subsets significantly increased from 0.36% to 38.58% and CD69 expression significantly increased from 0.11% to 83.74%.
Conclusion: The SEB-activated CD8(+);NKT cells proliferated in vitro and their cell division capability could be determined by CFSE staining and FCM.