8 pcpt camp

Ovary ECD assays were conducted in four saline solutions referred to as standard saline, nominally Ca²⁺ free saline, Ca²⁺ free saline, and high Ca²⁺ saline. Previous studies established that ovaries dissected from A. aegypti females sustain ECD production in vitro over multiple hours when cultured in standard saline (1.7 mM CaCl₂, 1 mM MgCl₂, 1.8 mM NaHCO₃, 3.4 mM KCl, 5 mM trehalose, 25 mM HEPES, 150 mM NaCl) (Riehle and Brown, 1999 (link)). Nominally Ca²⁺ free saline was standard saline without CaCl₂. Ca²⁺ free saline lacked CaCl₂ and contained EGTA (2 mM) while high Ca²⁺ saline contained 8.5 mM Ca²⁺. Other modifications to standard saline included the addition of 10 or 100 nM gadolinium chloride (GdCl₃) (Sigma G7532), 2 or 20 μM ionomycin (Sigma 10634), or 10 μM thapsigargin (Calbiochem 586005). Stocks of thapsigargin and ionomycin (2 mM) were prepared in ethanol while GdCl₃ (1 mM) was prepared in water. NaHCO₃ was replaced in standard saline with additional NaCl to 151.8 mM to avoid formation of insoluble Gd₂(CO₃)₃ when GdCl₃ was used at a working concentration of 100 nM. Preliminary experiments showed this modification in the absence of GdCl₃ had no effect on ovary ECD production. 8-pCPT-cAMP (Sigma C3912) was made as a 10 mM stock in water and added to saline solutions at 100 uM.