Were washed in phosphate buffer saline (PBS, pH 7.4) and counted in
Were washed in phosphate buffer saline (PBS, pH 7.4) and counted in a haemocytometer by trypan blue exclusion method. Treatment schedule of CuNG and Dox CuNG was injected once i.p. at a dose of 10 mg/kg body weight [dissolved in DMSO and finally suspended in normal saline to achieve final concentration of 0.1 (v/v) DMSO in normal saline]. Untreated control group received i.p. injection of 1 ml vehicle (i.e., 0.1 (v/v) DMSO in normal saline) only. In EAC/Dox bearing mice, CuNG was administered 7 days after inoculation with EAC/Dox. Previous work from our laboratory had showed that at a dose of 10 mg/kg body weight CuNG had no toxicity [15]. Animals were euthanasized 2 h or 24 h after CuNG treatment unless mentioned otherwise. Measurement of glutathione (GSH) GSH was measured following the method of Sedlack and Lindsay [19]. Briefly, tissue/1 ?106 cell homogenate in 0.Page 2 of(page number not for citation purposes)BMC Cancer 2006, 6:http://www.biomedcentral.com/1471-2407/6/ml PBS was mixed with 2.4 ml EDTA (0.02 M), 2 ml deionized water and 0.5 ml 50 TCA and centrifuged at 500 ?g for 15 min at 4 . 2 ml of supernatant was mixed with 2 ml 0.4 M Tris buffer (pH 8.9). 50 l 5,5′-dithio bis (2-nitrobenzoic acid) [DTNB] (0.01 M) was added to the mixture. Within 2? min of addition of DTNB, optical density (OD) was measured at 412 nm. Protein was measured by using the Bradford Method [20].Measurement of glutathione peroxidase (GPx) activity GPx activity was measured from the tissue homogenate. Tissue homogenate was prepared following the method of Hafemann et al [21]. In brief, the animals were sacrificed, the organs were dissected, dried and weighed. The homogenate was prepared with 0.15 M KCl solution and centrifuged at 10,000 rpm for 20 min at 4 . The supernatant was analysed according to the reported method [21,22]. Measurement of catalase (CAT) Catalase was measured by the reported method [21,23]. In brief, tissue homogenate was prepared using 0.1 M phosphate buffer solution (PBS, pH7.4) and centrifuged at 1,00,000 g for 1 h at 4 . Tissue homogenate was transferred in 0.1 M PBS (pH 7.4) containing 0.45 M H2O2. Aliquots of the mixture (0.5 ml) were removed at 20 S intervals and added to 2.0 ml of solutions containing 0.2 mg/ml O-dianisidine, 0.015 mg/ml peroxidase and 0.81 mg/ml sodium azide. After 10-min incubation at room temperature, 50 H2SO4 solution was added to stop the reaction. The absorbance of the reaction mixture was measured at 530 nm. One unit of enzyme activity (k) was calculated as follows:Measurement of serum copper level For serum collection, mice were anesthetized and their chests were cleaned with ethanol. Blood was obtained via closed cardiac puncture by means of a 22-guage hypodermic needle [25]. Blood from each group (CuNG treated and untreated; normal and EAC/Dox bearing mice) was taken in separate glass tubes, clotted, chilled to 4 and centrifuged for 20 min at 3,000 rpm. Serum was removed, immediately filtered (0.22 m) and stored at 4 (if used within 24 h) or frozen. To 100 l serum taken in a test tube, 3.9 ml of nitric acid (2.5 ) was added and vortexed for 5 min. The solutions were kept at 37 incubator for 6 h with occasional shaking. The mixture was centrifuged at 500 ?g for 5 min. Cu was measured in the clear supernatant in Flame Atomic Absorption Spectrophotometer (AAS) [Varian Spectra 200 FS, hollow cathode PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25609842 lamp, Flame type: Air acetylene; replicate 3; wavelength 324.8 nm]. Aviptadil web Measurements of copper in liver.