Flow cytometric measurement of neutral red accumulation in earthworm coelomocytes: Novel assay for studies on heavy metal exposure

Neutral red (NR) is a vital stain accumulating in lysosomes of the cells, exemplified here by the coelomocytes non-invasively extruded from the earthworm coelomic cavity. Flow cytometry allows to distinguish two main populations of earthworm coelomocytes, namely hyaline and granular amoebocytes (A), and eleocytes/chloragocytes (E), the latter exhibiting riboflavin-derived autofluorescence. Coelomocytes of Dendrobaena veneta, Eisenia fetida, Allolobophora chlorotica, and Lumbricus sp. were incubated in vitro with copper chloride at 0 (controls) to 88 μg/ml. Both amoebocytes and eleocytes accumulated neutral red, as evidenced by light and confocal microscopy and by flow cytometry. Flow cytometry allowed quantitative analysis of the adverse effects of an in vitro copper exposure on the intracellular neutral red accumulation in coelomocytes, phenomenon being completely prevented in the presence of metal-chelating agent, ethylenediaminetetraacetic acid (EDTA). In conclusion, flow cytometry may be used as an objective and fast tool for the measurement of NR accumulation in invertebrate cells, and the neutral red retention in the cells subjected to various stressors, including environmental pollution.