2 nM, did not show differences

in total adhering hemocyte counts [CTX concentrations (nM): 0.0: 396.7±8.9 cells/mm2; 1.2: 398.8±8.9 cells/mm2; 6.0: 412.8±9.0 cells/mm2; 12.0: 400.7±8.9 cells/mm2; 30.0: 412.8±9.0 cells/mm2; 60.0: 369.1±8.6 cells/mm2; 120.0: 419.5±9.2 cells/mm2; p>0.05]. Hence the drop in total hemocytes at 1.2 nM CTX was not due to hemocyte detachment or lysis. Non-attached hemocytes ( Fig. 2E) collected from unwashed monolayers treated with 1.2 nM CTX were predominantly aggregated hemocytes (individual: 0 cells/mm2; aggregated: 93.5±2.7 cells/mm2). The number of non-attached and adhering cells determined from slide areas with hemocytes treated with 1.2 nM CTX were combined, and the resulting total sum was not significantly different (p>0.05) from the sum of adhering (individual and aggregated) hemocytes in the PBS control group, or higher CTX LY2109761 manufacturer concentrations. Thus decline in counts at 1.2 nM CTX was due to the initial inhibition of hemocyte adhesion as opposed to hemocyte detachment or lysis. A decline in individually adhered hemocytes from 6 to 60 nM CTX negatively correlates with the increase in hemocytes in microaggregates (R=−0.97; p<0.05), implying, with the absence of lysis and detachment, that hemocytes aggregate when

treated with high, albeit still at concentrations of CTX below Fluorouracil supplier traditionally used levels. Adding the sum of individual hemocytes and aggregated cells produced the same pattern and magnitude as did CTX (p>0.05) including the nonlytic decline at 1.2 nM CTX. Thus CTB is responsible for much of the CTX effect on hemocyte adhesion. Since soluble RGDS peptides inhibit integrin-mediated hemocyte encapsulation of Sephadex beads in Chrysodeixis (Pseudoplusia) includens [53], soluble RGDS tetrapeptides were used to determine if CTX-induced in vitro microaggregation might be elicited by integrins. CTX concentrations 1.2 and 120 nM produce comparable increases in total microaggregation size and frequency in vitro (p>0.05) ( Fig. 2A) and similar responses to RGDS/RGES treatments thus 1.2 nM CTX is presented showing www.selleck.co.jp/products/DAPT-GSI-IX.html the effects of RGDS on hemocyte aggregation.

Five mM soluble RGDS significantly inhibited the total aggregated hemocyte area by 50% (27.5–32.1; p<0.05) and microaggregate frequency by 50% (17.0–27.4; p<0.05) at both CTX concentrations compared with the absence of the tetrapeptide, whereas RGES had no effect ( Fig. 3A for 1.2 nM CTX). The decline in both total aggregated hemocyte area and microaggregate frequency did not reflect RGDS-induced hemocyte lysis or detachment because RGDS did not decrease the number of attached hemocytes on preformed monolayers after 30 min incubation (total hemocyte counts from PBS control: 412±9.1 cells/mm2; 5mM RGDS: 401±8.9 cells/mm2; p>0.05). Hemocytes treated with PBS only showed less pronounced aggregation ( Fig. 3B) than CTX-treated hemocytes ( Fig.