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Stage-Dependent Accumulation of Cadmium: MATERIALS AND METHODS(5)

MATERIALS AND METHODS(5)

Subcellular 109Cd Distribution

To determine the subcellular distribution of 109Cd in testes, like stages (GZ/PrM, M, PoM) were pooled from animals injected i.v. with a tracer dose of 109Cd 3 days (experiment 1) or 7 days (experiment 2) previously. Kidney was taken as control. Tissues were homogenized and cellular subfractions prepared using buffers and procedures essentially as described for characterizing steroid receptors in cytosolic and nuclear extracts of shark testis, with the exception that monothioglycerol was omitted from all buffers to avoid possible interference with Cd. Tissue aliquots (0.3-3.0 g) were homogenized in buffer H (1:3 w:v; 50 mM Tris HCl, 1 mM EDTA, 30% glycerol; pH 7.5) using a Polytron (Brinkman, Westbury, NY; three 15-sec bursts) and centrifuged at 1000 X g for l5 min to obtain a crude nuclear subfraction. buy yasmin online

The pellet was washed three times with buffer W (10 mM Tris HCl, 3 mM MgCl2, 0.25 M sucrose; pH 7.5), each time followed by centrifugation at 1000 X g for 15 min, and then extracted for 60 min with buffer H containing 0.7 M KCl (buffer E, original vol) and centrifuged at 100 000 X g for 60 min to obtain a salt-extractable nuclear fraction. The remaining pellet was designated the salt-resistant nuclear fraction. The 1000 X g supernatant was centrifuged at 100 000 X g for 60 min to obtain a cytosolic extract, and the remaining pellet was designated membranes (plus mitochondria). Radioactivity was measured in triplicate aliquots of each fraction. Values were expressed as cpm/g tissue.