The FW crayfish molting cycle can be used as a model to study Ca transport mechanisms across epithelial membranes. The objective was to quantify the kinetics of ATP-dependent Ca uptake into BLMV from the crayfish kidney and liver using flow cytometry. Vesicles were isolated from individual animals at various stages in the molting cycle. They were loaded with the fluorescent indicator fluo-3 pentapotassium salt. The BLMV were frozen using methanol/dry ice and stored at -80C in a buffered 250 mM Trehalose solution, before shipment to HSS. The BLMV were slowly thawed and treated with polyclonal antifluorescein antibodies. Exposure of intermolt liver BLMV to free Ca in the presence of ATP resulted in little to no Ca uptake as compared to control (no free Ca); however premolt BLMV showed 35.4% increase in ATP-dependent uptake as compared to control and the postmolt BLMV showed a 35.1% decrease in ATP-dependent Ca uptake as compared to control. The intermolt kidney BLMV produced a 35.8% increase in ATP-dependent Ca uptake as compared to control while the premolt and postmolt produced 47.6% and 59.8% increase in ATP-dependent Ca uptake as compared to control, respectively. Synchronized molting is difficult to achieve in crayfish; therefore it is necessary to develop a sensitive assay that can be performed on small amounts of tissue from individual animals. These preliminary results would indicate that flow cytometry can be used to detect ATP-dependent Ca uptake into individual crayfish BLMV.
|Original language||English (US)|
|State||Published - Mar 20 1998|
ASJC Scopus subject areas
- Molecular Biology