A double staining flow cytometric assay for the detection of steroid induced apoptotic leucocytes in common carp ( Cyprinus carpio)

Abstract

Steroid hormones play an important role in the regulation of the immune system through different ways. In this in vitro study, the effects of steroid hormones on the apoptosis of leucocytes were evaluated to understand the involvement of this process in the immunocompetence of common carp. Prior to the investigation, a double staining flow cytometric assay using fluorescein diacetate (FDA), which reacts with esterases of viable cells, and propidium iodide (PI), an acid dye that binds with nuclear DNA, was established. FDA and PI negative cells were regarded as apoptotic. The FDA–PI technique is comparable to the Annexin V–PI technique and can be used in the quantification of the apoptosis of fish leucocytes accurately. The results suggest that the disappearance of esterases and externalization of phosphatidylserine (PS) may be common to many apoptotic pathways. Cells collected from peripheral blood, spleen, head kidney, and thymus were cultured for 16 h either in the absence or presence of steroid hormones, i.e. cortisol (F), testosterone, 11-ketotestosterone, and estradiol-17β, and analyzed by flow cytometry followed by the FDA–PI method. Results showed that F induced apoptosis in leucocytes from blood and other lymphoid organs suggesting the role of F as an immune regulator. The participation of sex steroids to the immunocompetence of carp was not found, since they did not induce apoptosis of leucocytes in any organ.