. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 260 CHEN AND C. J. BAYNE 1 90 iO V I 20. The inhibitory effects of caffeine, NEM. EDTA. ami cytochalasin B on hemocyte adhesion and spreading 0 5 10 15 20 Time of adhesion (mm) Figure 5. The influence of temperature on the adhesion of Myli/its californianm hemocytes in vitro Hemolymph was loaded into 96-well culture plates (50 ^I/well). These were then incubated at 4°C or 22 ± 1°C. At 5. 10, and 15 min, the non-adherent cells and plasma were removed. The wells were washed three times with CMTBS. and then the adherent c
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 260 CHEN AND C. J. BAYNE 1 90 iO V I 20. The inhibitory effects of caffeine, NEM. EDTA. ami cytochalasin B on hemocyte adhesion and spreading 0 5 10 15 20 Time of adhesion (mm) Figure 5. The influence of temperature on the adhesion of Myli/its californianm hemocytes in vitro Hemolymph was loaded into 96-well culture plates (50 ^I/well). These were then incubated at 4°C or 22 ± 1°C. At 5. 10, and 15 min, the non-adherent cells and plasma were removed. The wells were washed three times with CMTBS. and then the adherent cells were lysed for BCA protein assay. The protein values ob- tained for wells in which hemolymph was held at 22 ± 1°C for 15 min were used as 100% hemocyte adhesion. There is a significant difference (P < ) at 5 min incubation at 4°C, and at 22°C. no significant differences in hemocyte adhesion and ag- gregation when hemolymph was mixed (1:1) with CMTBS in the range of 800-1100 mOsm. The inhibitory effects of caffeine. NEM. EDTA, and cytochalasin B on cell aggregation Hemocytes exposed to 25 mM caffeine, 5 jug/ml cyto- chalasin B. or mM NEM were strongly inhibited from aggregating (95%, 71%, and 72%, respectively, remained free). When hemocytes were treated with EDTA, the morphology of aggregates varied. Under the microscope, some colorless material was seen among the aggregates. Its presence seemed to restrict hemocyte contact. Though caffeine inhibition at 25 mM or 50 mAf was reversible and rapid, it did not kill the cells. At a caffeine concen- tration of 15 mAf, cells formed weak aggregates. The dif- ference between the percent free cells in 25 mM and 15 mM caffeine (means of and ) was statisti- cally significant (P < ) (Fig. 7a). Cytochalasin B (5 /ig/ml) in DMSO resulted in 70% of the hemocytes remaining free. DMSO () did not influence hemocyte aggregation and adhesion (Fig. 7b). At mM, NEM sig- nificantly inhibited hemocy
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