Screening of in vitro Stimulation Methods for iNKT Cells and Conditions for in vitro Activation of iNKT Cells by Triptolide

doi:10.19803/j.1672-8629.20230800

Abstract

Abstract: Objective To study the methods of activating iNKT cells and the conditions for TP's activation of iNKT cells in vitro. Methods Flow cytometry was performed to detect the activation marker CD69 and cytokine IL-2 of iNKT cell line DN32.D3 cells using either a combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin (ION) or a combination of CD3e and CD28 antibodies. The liver nonparenchymal cells of mice were stimulated under different concentrations of PMA. The levels of CD69, IL-4, IL-17 and IL-10 in iNKT cells were detected by flow cytometry to screen the optimal methodological condition for the in vitro activation before the conditions for in vitro activation of DN32.D3 cells by TP were investigated. Results The best activation of iNKT cells was achieved when stimulated by the combination of PMA and ION with the concentration of PMA at 50 ng·mL^-1. TP activated DN32.D3 cells when antigen-presenting cell (APC) RBL-CD1d cells were added for co-culture. Conclusion INKT cells are most activated when the PMA concentration is 50 ng·mL^-1 with the combination of PMA and ION. TP activation of iNKT cells requires the presence of APCs.

Key words: Triptolide, Phorbol 12-Myristate 13-Acetate, Invariant Natural Killer T Cells (iNkT Cells), Liver, DN32.D3 Cells, in vitro Activation, Flow Cytometry, Mice

CLC Number:

R994.11

LIANG Xiaojing, CHEN Xin, NONG Cheng, LI Liang, DING Jian, ZHANG Luyong, JIANG Zhenzhou, WANG Xinzhi. Screening of in vitro Stimulation Methods for iNKT Cells and Conditions for in vitro Activation of iNKT Cells by Triptolide[J]. Chinese Journal of Pharmacovigilance, 2025, 22(2): 147-154.

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https://zgywjj.magtechjournal.com/EN/Y2025/V22/I2/147

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