After the final wash the hemocytes were lysed in 50 μl chilled 0.1% (v/v) Triton X-100 in 0.1 M HCl, vortexed (30 sec) and ultrasonicated (3 pulses, 5 s) ensuring complete lysis. Samples not used immediately were stored at −80 °C. Intracellular cAMP from a minimum of 5 replicates was determined using the acetylated version of cAMP immunoassay kit (Assay Designs, Plymouth Meeting, PA). One time point (30 min) was selected for the cAMP assay as elevated levels of intracellular

cAMP in hemocytes are sustained 30 min post-bacterial injection [45]. Data were see more analyzed using the 95% confidence limit overlap protocol [65]. Percentage data are recorded as the decoded mean with 95% confidence limits in 2 arcsin √p-transformation. Graphic and tabular data are presented as the mean±standard error of the mean. An a priori α value of 0.05 was chosen. The

number of individual hemocytes adhering to the slides decreased with increasing incubation time and increasing CTX concentrations (Fig. 1). However, the number of adhering total aggregated cells remained the same at 20 min independent of CTX concentration (p>0.05), but by 30 min incubation, as the total hemocyte counts decreased, the level of adhering total aggregated cells increased ( Fig. 1). The latter suggesting individual hemocytes engaged in hemocyte–hemocyte accretions until a critical microaggregate http://www.selleckchem.com/products/MK-1775.html size is achieved, explaining the low levels of initial total aggregated cells despite decreasing total hemocyte counts. In order to further assess the hemocyte aggregation induced by CTX; all subsequent in vitro reactions were incubated for 30 min. The concentration of glass-attached individual and aggregated hemocytes was determined with a broader range of CTX concentrations. The total of individual hemocytes during exposure to 1.2 nM CTX significantly dropped (45%; 25.9–27.5; p<0.05; Fig. 2A) compared with the PBS control. At

6 nM CTX adhesion significantly increased (82%; 50.7–60.9; p<0.05) above 1.2 nM CTX levels and subsequently decreased linearly with increasing 17-DMAG (Alvespimycin) HCl CTX concentration (r2=−0.93; p<0.05) to a maximum decline of 37% (15.2–15.9) at 60 nM CTX which was 65% (44.9–56.7) below the PBS control ( Fig. 2A). Levels of individually attached hemocytes plateaued at 120 nM CTX ( Fig. 2A). Granular cells and plasmatocytes adhered to the same extent (p<0.05) in the PBS control and significantly decreased to similar levels (p<0.05) at 1.2 nM CTX. Granular cell and plasmatocyte attachment increased at 6 nM CTX, levels of adhering granular cells being greater (15%; 8.5–8.9) than levels of plasmatocytes at this CTX concentration. Attachment of granular cells and plasmatocytes decreased linearly from 6 nM to 120 nM CTX by 23% (13.5–14.2) and 30% (16.8–17.7), respectively. In contrast, the sum total of aggregated cells (total hemocytes in all aggregates) increased above the PBS control values at 1.2 nM CTX, followed by a significant decrease (p<0.05) at 6 nM ( Fig. 2A).