Assessment of the Cell Viability of Cultured Perkinsus marinus (Perkinsea), a Parasitic Protozoan of the Eastern Oyster, Crassostrea virginica, Using SYBRgreen–Propidium Iodide Double Staining and Flow Cytometry

Abstract

A flow cytometry (FCM) assay using SYBRgreen and propidium iodide double staining was tested to assess viability and morphological parameters of Perkinsus marinus under different cold- and heat-shock treatments and at different growth phases. P. marinus meront cells, cultivated at 28 degrees C, were incubated in triplicate for 30 min at -80 degrees C, -20 degrees C, 5 degrees C, and 20 degrees C for cold-shock treatments and at 32 degrees C, 36 degrees C, 40 degrees C, 44 degrees C, 48 degrees C, 52 degrees C, and 60 degrees C for heat-shock treatments. A slight and significant decrease in percentage of viable cells (PVC), from 93.6% to 92.7%, was observed at -20 degrees C and the lowest PVC was obtained at -80 degrees C (54.0%). After 30 min of heat shocks at 40 degrees C and 44 degrees C, PVC decreased slightly but significantly compared to cells maintained at 28 degrees C. When cells were heat shocked at 48 degrees C, 52 degrees C, and 60 degrees C heavy mortality occurred and PVC decreased to 33.8%, 8.0%, and 3.4%, respectively. No change in cell complexity and size was noted until cells were heat shocked at >or=44 degrees C. High cell mortality was detected at stationary phase of P. marinus cell culture. Cell viability dropped below 40% in 28-day-old cultures and ranged 11-25% in 38 to 47-day-old cultures. Results suggest that FCM could be a useful tool for determining viability of cultured P. marinus cells.

Heat shock protein (hsp70) expression and thermal tolerance in sublethally heat-shocked eastern oysters Crassostrea virginica infected with the parasite Perkinsus marinus

“…The sublethal heat shock temperature also may have affected function in P. marinus. Heat treatments of 40°C and higher for 30 min reduced the viability of cultured P. marinus (Soudant et al 2005). High temperatures that oysters are capable of withstanding may limit the growth of P. marinus.…”

Effects of temperature and salinity on the survival of Bonamia ostreae, a parasite infecting flat oysters Ostrea edulis

“…Flow cytometry has been successfully used to measure cell viability of cultured Perkinsus marinus, a parasitic protozoan of the eastern oyster Crassostrea virginica (Soudant et al 2005). This tool allows multiparametric analyses on a large number of cells in a very short time and thus presents advantages over microscopic approaches.…”

Cord Blood Mononuclear Cells Have a Potential to Produce NK Cells Using IL2Rg Cytokines

“…18,19 IL-2 is the firstly identified member of IL2rg family, and its gene was originally cloned on the basis of the T-cell growth factor activity of this cytokine. 20,21 Besides its T cell growth factor activity, IL-2 up-regulates NK cell proliferation and function, induces lymphokine-activated killer (LAK) activity, and also mediates activated B cell proliferation and Ig production. 21,22 Umbilical cord blood is an easy access sources to use in transplantation and immunotherapy.…”

“…20,21 Besides its T cell growth factor activity, IL-2 up-regulates NK cell proliferation and function, induces lymphokine-activated killer (LAK) activity, and also mediates activated B cell proliferation and Ig production. 21,22 Umbilical cord blood is an easy access sources to use in transplantation and immunotherapy. Probably NK cell could be geenerate from cord blood cells.…”