Pharmacology & Toxicology Poster Session
Materials & Methods
Discussion & Conclusion
the Role of Divalent Cations in the Mechanisms of EDTA Cytotoxicity
Contact Person: Michael O. Ogundele (firstname.lastname@example.org)
Materials and Methods
Reagents and Buffers
Gelatin Veronal buffered saline (GVB2+) containing 0.15mM of Ca2+ and 1mM of Mg2+ were prepared as described (Mayer 1971). In addition GVB-- and GVB-- containing varying levels of calcium and magnesium ions in the form of their chloride salts, up to 50mM of each were also prepared.
Rabbit red blood cells and the hemolytic assay
Non-sensitized rabbit erythrocytes (ORBO 20/21) were obtained from Behringwerke, Marburg, Germany. Using a micro-modification of the standard complement hemolytic assay, 40µl of 5 x 108 rabbit red blood cells/ml were added to Veronal buffer containing a final EDTA concentration of 10mM and different concentrations of calcium and magnesium ions, to obtain a total reaction volume of 400µl with different volumes of samples to be tested. The reaction mixture was incubated for 60 minutes at 37°C, with occasional mixing. The reaction was stopped by addition of 1ml of cold GVB2- and the cells were pelleted by centrifugation at 12000g for 5 minutes. 200µl of each supernatant was pipetted into a 96 well flat microtitre plate (Greiner, Frickenhausen, Germany) and the amount of released hemoglobin recorded by a plate spectrophotometer (MR 700 microplate reader, Dynatech Instruments, Inc. Torrance, California) at a test wavelength of 402 against a reference wavelength of 612.
From the values of optical densities (OD) obtained, the degree of haemolysis produced by a given volume of HBM was obtained from the equation:
Y = Test OD - spontaneous lysisOD - HBM blank OD X 100
H2O lysis OD - spontaneous lysis OD
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