Tehran University Medical Journal (Nov 2002)
"The adherence and functional properties of mice peritoneal macrophage phagocytic system with Phorbol Myristate Acetate stimulation and Nitroblue Tetrazolium reduction assay "
Abstract
Introduction: PMA is known to induce the differentiation of monocytes to macrophages. This agent also increases the killing effect of the monocytes/macrophages through oxidative burst and can be used as a stimulant for oxidative burst assay. The present experimental study was intended to investigate the in vitro effects of PMA on the differentiation, morphological changes, cell adherence and the viability of monocyted-derived macrophages (MDMs). Besides, MDM capacity for free radical production was assessed, indicating the oxidative burst events. Materials and Methods: This experimental study has been design in Department of Immunology of S.B.M.U in Tehran Iran (year 2000). Peripheral mononuclear cells from adult Balb/c mice were isolated and cultured in complete tissue culture medium and divided in two group: control, (without PMA) and test (were added Pma=450 ng/ml). MDMs wee counted on the hours 1, 2, 3, 4, 6 and 18 and their characteristics were confirmed by morphological analysis (histological features) in both groups. Viability of MDMs was assessed using trypan blue. In the peak time of MDMs activation the oxidative burst was determined by NBT reduction. Results: The obtained results suggested that PMA had significant effect on the differentiation of monocytes to macrophages. The morphologic maturation tended to occur in earlier stages in the PMA treated cells comparing to the control MDMs. Also, the number of adherent cells was considerably more in PMA stimulated monocytes. The peak time of cell adherence in the presence of PMA was no the second hour. As the incubation period increased, the significant difference between the numbers of adherent cells in two culture systems decreased. However, viability decreased significantly in the PMA treated MDMs, i.e. PMA treatment induced rapid apoptosis in the MDMs after their activation. PMA stimulated MDMs markedly (60%). Also we mentioned that the primary un-stimulated MDMs only revealed (55%) of NBT reduction after treatment with PMA at NBT reduction stage. Conclusion: Phagocytic function and oxidative burst assay in monocyte-macrophage lineage can be a diagnostic tool for identification and management of some Immunological abnormality and defect and can be establish distinct from other phagocytic system assessment.
