Objective: The aim of the present study was to evaluate the surface expression of moesin on differentiated macrophage-like THP-1 cell surface upon LPS stimulation.
Methods: THP-1 cells were cultured and treated with 200nM phorbol myristate acetate for 18 hours to induce differentiation into macrophage-like cells. Adherent cells (differentiated cells) were incubated for different periods with several concentrations of E. coli LPS. Cells were then fixed with cold methanol for 5 minutes at -10 degrees C. For membrane permeabilization, cells were incubated with 0.25% Triton X-100 in PBS for 20 minutes at room temperature. Nonspecific binding was blocked by 10% normal blocking serum in PBS for 30 minutes. The cells were incubated with either moesin antibody or relevant IgG as control for 60 minutes followed by 45 minutes incubation with FITC-conjugated secondary antibody. After mounting, the slides were examined using fluorescence microscope.
Results: There was positive and similar expression of moesin on the cell surface of both permeabilized and non-permeabilized cells. Upon LPS stimulation (10ng/ml) fluorescent intensity peaked after 5 minutes. Fluorescent intensity plateaued for 2 hours after which it began to decay.
Conclusions: These findings suggest that moesin is expressed on the surface of differentiated macrophage-like THP-1 cells. The expression increased upon LPS stimulation. This up-regulation may be due to either a conformational change in the moesin molecule or recruitment of moesin to the cell surface. Supported by USPHS Grant DE13191.