The cytotoxicity of phorbol 12- myristate 13-acetate and lipopolysaccharide on THP-1 cells and optimized differentiation protocol

In order to evaluate the immunomodulatory potential of novel monofloral Irish honeys, THP-1 monocyte-derived macrophages provided a suitable cell-based model. THP-1 cells can be differentiated to macrophages using phorbol-12-myristate-13-acetate (PMA). Differentiated cells are then challenged with lipopolysaccharide (LPS) to stimulate the inflammatory cascade. Few studies on the cytotoxic concentrations of these two compounds on THP-1 cells are published. Therefore, the viability of treated THP-1 cells was initially evaluated using trypan blue dye exclusion (PMA), the resazurin assay (LPS), and propidium iodide staining. Furthermore, research to date suggests concentrations ranging from 100 ng/ml down to 15 ng/ml (Lund et al., 2016) and 5 ng/ml (Park et al., 2007) PMA are sufficient for THP-1 differentiation. Consequently, the differentiation potential of this sub-cytotoxic PMA concentration range was also evaluated using flow cytometric detection of the macrophage-specific CD-14 cell surface marker.

The highest concentration of PMA (1 µg/ml) evaluated with the trypan blue exclusion assay caused ~20% cell death. Flow cytometry results indicated CD14 percentage for THP-1 cells exposed to 100ng/ml PMA for 48 hours (~14.63%, SD ± 3.94) were lower than 15ng/ml (~43.41%, SD ± 5.46) and 5ng/ml (~38.36%, SD ±13.12). No substantial time-dependent difference for these concentrations was observed following 48 versus 72-hour exposure. The highest LPS concentration, 100-fold higher than concentrations used in the literature, caused ~29.21% (resazurin assay) and ~25.76% (PI staining) cell death in differentiated THP-1 cells.

In summary, the differentiation capacity of THP-1 cells, and the differentiation ability of the test compounds require cytotoxicity assays to be chosen carefully. Important variables affecting assay outcomes include phenotypic differences between untreated control cells (monocytes) versus treated monocyte-derived macrophages in terms of metabolic and pinocytosis rates.