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Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes
Published on: May 31, 2018
[Effects of heat shock protein gp96 on function of macrophages from mouse]
Tian-Yi Zhang1, Hua Wang, Xin-Yang Zhou
1Laboratory of Analytical Cytology, Jiangsu Provincial Key Laboratory of Neuroregeneration, Nantong University, Nantong, Jiangsu 226001, P. R. China.
Background & Objective:
Heat shock protein gp96 (HSPgp96) plays an important role in antigen presenting and specific antitumor immunotherapy, but its effects on macrophages need further investigation. This study was to investigate the effects of HSPgp96 derived from H22 tumor cells on mouse peritoneal elicited macrophages (PEMphi) by detecting some factors that are related to the function of the macrophages.
Methods:
After macrophages were stimulated by HSPgp96, dynamic changes of intracellular free calcium (Ca2+), nitric oxide (NO) and reactive oxygen species (ROS) in the macrophages were measured by the laser scanning confocal microscopy (LSCM) with sensitive fluorescent dye Fluo-3/AM, DAF-FM-DA, and H2DCF-DA. The color immunofluorescence assay was used to observe the expression intensity and distribution of MHC II and CD86 in the macrophages.
Results:
The production of Ca2+, NO and ROS increased rapidly in the macrophages after stimulation of HSPgp96. Their concentration peaks appeared at 30 s, 80 s, and 580 s after stimulation with the increase scopes of (161.06+/-70.99)%, (77.58+/-31.17)%, and (647.28+/-185.70)%, respectively. The positive rates of MHC II and CD86 were significantly higher in 60 mg/L HSPgp96-stimulated macrophages than in control macrophages [(61.8+/-5.1)% vs. (40.9+/-3.5)%, P<0.01; (54.9+/-2.0)% vs.(23.1+/-3.1)%, P<0.01].
Conclusion:
The in vitro stimulation of HSPgp96 may enhance the production of Ca2+, NO and ROS in mouse PEMphi, and up-regulate the expression of MHC II and CD86.
