Flow cytometric quantification of proliferating coelomocytes non-invasively retrieved from the earthworm, Dendrobaena veneta

Earthworms irritated naturally (e.g. by predators) or experimentally extrude coelomocyte-containing coelomic fluid through the dorsal pores of the body wall. In the present study, the earthworms, Dendrobaena veneta, experimentally depleted of free-floating coelomocytes by multiple electric shocks (1 min, 4.5 V) remained fully vital and coelomocyte depletion was followed by the extensive cell replenishment, which was more efficient in the case of amoebocytes than autofluorescent eleocytes/chloragocytes, quantified by flow cytometry. Immunohistochemical procedure with antibodies against human Ki-67 proliferation antigens revealed proliferating cells on cytospin preparations of coelomocytes extruded by electric shock. Quantification of proliferating cells in the suspension of extruded coelomocytes was performed by flow cytometry on FL-2 profiles of propidium iodide-stained samples; riboflavin-derived autofluorescence of eleocytes/chloragocytes was lost during detergent treatment. As expected, the percentage of coelomocytes proliferating in coelomic fluid was increased during restoration of coelomocyte number after experimental depletion. The method described here may be very useful for investigations of antigen-driven proliferation of earthworm coelomocytes.