Flow cytometry methodology for measuring chemical induced cellular dead cell apoptosis and necrosis of splenic leucocytes in European bullhead, Cottus sp

In environmental immunotoxicology, leucocyte apoptosis and necrosis parameters may constitute pertinent biomarkers of immunological disturbance due to their modulations by numerous xenobiotics. In an ecotoxicological concern, the aim of this study was therefore to develop immune-related biomarkers using flow cytometry methodology in the European bullhead, Cottus sp., a current freshwater fish. In this context, spleens of bullhead were interesting due to sampling facilities and to their immune function. After spleen tissue disruption, leucocytes of each organ were isolated using density gradient centrifugation and cells were adjusted to 106 cells.mL-1. For each leucocyte sample, five aliquots were carried out. With the first aliquot, autofluorescence of cells were detected. YO PROR-1 (100 uM) and Propidium Iodide (PI, 1.5 mM) were added in two different aliquots in order to obtain cellular fluorescence parameters indicating the presence of apoptotic and necrotic leucocytes, respectively. Both YO-PROR-1 and PI were added in a double markedly method. Spleen leucocyte cellular aliquots were also treated with YO-PROR-1, PI and quinoline alkaloid camptothecin (100 uM) which induce apoptosis. After incubation, cellular analysis were carried out with a CyanTM ADP flow cytometer connected to hypercytRintellicyt (Beckmann Coulter). Apoptotic and necrotic dead cells showed green and red fluorescence, respectively. The camptothecin treated cells presented similar necrotic values and a higher percentage of apoptotic cells than those observed in controls. In consideration of these developments, dead cell apoptosis and necrosis were tested during in vivo controlled-laboratory experiments where European bullheads naturally infected with Saprolegnia sp. and Ichthyophtirius sp. were exposed to 25 micro g.mL-1 of Copper. This exposure induced a decrease of cellular apoptosis and necrosis in contaminated organisms compared to controls. In conclusion, this work enabled a technical methodology to evaluate fish immune-physiological markers on spleen leucocyte of the sedentary bullhead, Cottus sp. For the assessment of wild fish health, further studies will be undertaken in order to characterize the potential and to validate the interest of these biomarkers in immunotoxicology studies