Development of a flow cytometric, non-radioactive cytotoxicity assay in Eisenia fetida: An in vitro system designed to analyze immunosuppression of natural killer-like coelomocytes in response to 7, 12 dimethylbenz[a]anthracene (DMBA)

We have developed a non-radioactive cytotoxicity assay to measure cytotoxicity mediated by natural killer (NK)-like effectors in the earthworm Eisenia fetida using a two-color fluorescence assay. K562, the target cell population, was labeled with 3,3′-dioctadecyloxacarbocyanine (DiO), a green fluorescent dye that stains the plasma membrane. NK-like coelomocytes isolated from E. fetida were used as the effector cell population. DiO-labeled targets were incubated with effector cells and the membrane impermeable, red fluorescent dye, propidium iodide (PI). PI labels the double-stranded nucleic acids in membrane-damaged cells only. Killed targets were analyzed using flow cytometry and exhibited coincident green membranes (DiO) and red-nuclear staining (PI). This methodology was employed to analyze the immunosuppressive effects of 7,12 dimethylbenz[a]anthracene (DMBA), a polycyclic aromatic hydrocarbon, on earthworm coelomocytes with NK-like activity. Flow cytometry and WinList™ Interative N-Color Compensation software revealed a significant decrease in the ability of coelomocytes to kill targets following in vitro DMBA exposure, compared to untreated controls where coelomocytes were cultured in the same medium in the absence of DMBA. These experiments demonstrate how flow cytometry can be used to measure cytotoxicity by earthworm coelomocytes cultured and exposed to DMBA in vitro to study the immunosuppressive effects of DMBA on NK-like effector cells of E. fetida.