. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. AMEBOCYTE AGGREGATION IN LIMULl'S 559 by preventing release of the aggregation promoting material from amebocytes, several approaches were employed. First, an attempt was made to isolate the active material from amebocytes treated with EDTA. Hemolymph was withdrawn into 100 mM EDTA so that the final concentration with amebocytes was 75 mM. After separation of the amebocytes by centrifugation, the EDTA—plasma was drawn off and the amebocyte pellet was washed and AHS prepared. The super- natant of this homogenate produces reve
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. AMEBOCYTE AGGREGATION IN LIMULl'S 559 by preventing release of the aggregation promoting material from amebocytes, several approaches were employed. First, an attempt was made to isolate the active material from amebocytes treated with EDTA. Hemolymph was withdrawn into 100 mM EDTA so that the final concentration with amebocytes was 75 mM. After separation of the amebocytes by centrifugation, the EDTA—plasma was drawn off and the amebocyte pellet was washed and AHS prepared. The super- natant of this homogenate produces reversal of EDTA inhibition, and despite an extended lag period, the aggregation is complete. From these results it appears that amebocvtes in EDTA do contain the APM. 100-1. (2) FIGURE 6. Tracings of representative graphs showing aggregation of EDTA inhibited amebocytes. Graphs are modified to smooth out oscillations; control aggregation in saline (1); amebocytes in 17 ± 6 HIM EDTA (2) ; amebocytes withdrawn in EDTA and resuspended in cell free plasma (3) ; amebocytes withdrawn in EDTA, resuspended in cell free plasma, and added to 1 : 20 dilution of serum (4) ; amebocytes in EDTA added to 1 : 20 dilution of AHS (5). A second approach employed to investigate possible inhibitory effects of EDTA on the release of APM from amebocytes involved direct assay of the supernatants of EDTA treated amebocytes. The ability of these supernatants to restore aggre- gation to amebocytes in EDTA is compared to that of the control supernatants of amebocytes treated with saline. Hemolymph is withdrawn in a 1:3 ratio with 100 mM EDTA, mixed and the suspension divided into equal aliquots in poly- ethylene centrifuge tubes. After the amebocytes are separated by centrifugation. the EDTA-hemocyanin supernatant is carefully removed, and the pellet of EDTA treated amebocytes covered with ml cell free plasma and ml physiological saline at room temperature. A total of 4 min elapsed between the tim
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Keywords: ., bookauthorlilliefrankrat, booksubjectbiology, booksubjectzoology
