APPENDIX Heparin 2 mg heparin was dissolved in 0.9 % NaCl (10 ml). 200 µl of heparin was added to each 1 ml of blood to prevent coagulation.
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1 APPENDIX 1 Preparation of reagents 1.1. Preparation of dosing solution Nonylphenol 15 mg of Nonylphenol was dissolved in olive oil (10 ml) and used as stock solution. The stock solution was serially diluted and used for treatments Heparin 2 mg heparin was dissolved in 0.9 % NaCl (10 ml). 200 µl of heparin was added to each 1 ml of blood to prevent coagulation Determination of protein Alkaline copper reagent Reagent A: 2 g of sodium carbonate was dissolved and made up to 100 ml with 0.1 N sodium hydroxide solution. Reagent B: 5 mg of copper sulphate was dissolved in 1.0 ml of 4 % sodium potassium tartrate. Reagent A of 50 ml and reagent B of 1.0 ml were mixed fresh at the time of use Sodium hydroxide (0.1 N) 0.4 g of sodium hydroxide was dissolved and made up to 100 ml with distilled water Sodium potassium tartrate (4%) 4 g of sodium potassium tartrate was dissolved and made up to 100 ml with Protein standard solution 100 mg bovine serum albumin (BSA) was dissolved and made up to 100 ml with This solution was diluted 10 times to obtain concentration of 0.1 mg/ ml Folin-Ciocalteau reagent (1 N) Commercial Folin-Ciocalteau reagent was diluted with two volume of distilled water. 153
2 1.4. Superoxide dismutase Tris HCl buffer (50 mm) containing EDTA (1 mm; ph 8.2) 605 mg of Tris HCl was dissolved in 100 ml of To this, g of EDTA was added and ph was adjusted to Pyrogallol (0.2 mm) in 50 ml of HCl (10 mm) 1.26 mg of pyrogallol was dissolved in 50 ml of 10 mm HCl HCl (10 mm) 41.6 µl of concentrated HCl was made up to 50 ml with distilled water 1.5. Catalase Phosphate buffer (0.05 M, ph 7.0) Solution A: 0.89 g of disodium hydrogen phosphate was dissolved in 100 ml of Solution B: 0.69 g of sodium dihydrogen phosphate was dissolved in 100 ml of 39 ml of Solution A and 61 ml of Solution B was mixed and made up to 200 ml with The ph was adjusted to H 2 O 2 (0.019 M) 58 µl of hydrogen peroxide (from 30 %) was made up to 100 ml with distilled water. Stored in cool and dark place Glutathione peroxidase Phosphate buffer (100 mm, ph 7.6) Solution A: 1.78 g of disodium hydrogen phosphate was dissolved in 100 ml of Solution B: 1.38 g of sodium dihydrogen phosphate was dissolved in 100 ml of 13 ml of Solution A and 87 ml of Solution B was mixed and made upto 200 ml with The ph was adjusted to EDTA (0.01M) mg of EDTA was dissolved in 10 ml of distilled water Sodium azide 154
3 6.5 mg of sodium azide was dissolved in 10 ml of Glutathione reductase mg of glutathione reductase was dissolved in 5 ml of Glutathione reduced mg of glutathione (reduced) was dissolved in 10 ml of NADPH (0.2 µm) mg of NADPH was dissolved in 10 ml of distilled water 1.7. Glutathione reductase Phosphate buffer (0.1 M, ph 7.6) Solution A: 1.78 g of disodium hydrogen phosphate was dissolved in 100 ml of Solution B: 1.38 g of sodium dihydrogen phosphate was dissolved in 100 ml of 13 ml of Solution A and 87 ml of Solution B was mixed and made upto 200 ml with The ph was adjusted to NADPH (0.2 µm) mg of NADPH was dissolved in 10 ml of Glutathione oxidized (2 mm) mg of glutathione oxidized was dissolved in 1 ml of EDTA (0.01 M) mg of EDTA was dissolved in 10 ml of 1.8. Hydrogen peroxide generation Phosphate buffer (0.05 M, ph 7.6) Solution A: 0.89 g of disodium hydrogen phosphate was dissolved in 100 ml of Solution B: 0.69 g of sodium dihydrogen phosphate was dissolved in 100 ml of 13 ml of Solution A and 87 ml of Solution B was mixed and made upto 200 ml with The ph was adjusted to
4 Horseradish peroxidase (8 units/ mg) 1 mg of horseradish peroxidase was dissolved in 10 ml of distilled water to make 8 units / ml Dextrose (100 nm) mg/ ml was prepared as a stock solution (1 M) and from this 100 nm was prepared by serial dilution with the distilled water Sodium hydroxide (10 N) 40 g of sodium hydroxide was dissolved in 100 ml of Phenol red (28 nm) mg/ ml was prepared as a stock solution (1 M) and from this 28 nm was prepared by serial dilution with the 1.9. Lipid peroxidation Trichloroacetic acid (TCA; 15%) 15 ml of TCA from 100% was made up to 100 ml with Thiobarbituric acid (TBA; 0.37%) mg of TBA was dissolved in 100 ml of HCl (0.25 N) 1.08 ml of HCl was made up to 50 ml with TCA: TBA: HCl (1:1:1) Equal volumes of the above three solutions were mixed to make 1: 1: 1 ratio of TCA: TBA: HCl Liver Transaminase assay Phosphate buffer (0.1 M, ph 7.4 Solution A: 1.78 g of disodium hydrogen phosphate was dissolved in 100 ml of Solution B: 1.38 g of sodium dihydrogen phosphate was dissolved in 100 ml of 13 ml of Solution A and 87 ml of Solution B was mixed and made upto 200 ml with The ph was adjusted to
5 Sodium pyruvate standard Sodium pyruvate (22 mg) was dissolved in 100 ml of phosphate buffer Sodium hydroxide 1.6 g of sodium hydroxide was dissolved in 100ml of AST substrate Dissolved 1.33 g of DL-aspartate and 15 mg of alpha-ketoglutatrate in dissolved. It was made upto 50 ml with 1 N NaOH. Adjusted ph to ALT substrate Dissolved 29.2 mg of alpha-ketoglutarate (200mM/L) and 1.78 g of DLalanine 2 mm/ L. it was made upto 50 ml with 1 N NaOH. Adjusted ph to ,4-Dinitrophenyl hydrazine Dissolved 19.8 mg of 2, 4-Dinitrophenyl hydrazine in 100 ml of 1 N HCl Hexokinase Triethanolamine buffer (0.05 M; ph 7.6) 450 mg of triethanolamine was dissolved in 50 ml of distilled water and the ph was adjusted to D-glucose (0.555 M) g of D-glucose was dissolved in 20 ml of MgCl 2 (0.1 M) 200 mg of MgCl 2 was dissolved in 10 ml of NADP (0.014 M) 117 mg of NADP was dissolved in 10 ml of ATP (0.019 M) 104 mg of ATP was dissolved in 10 ml with Phosphofructokinase Tris HCl buffer (50 mm; ph 8) 157
6 605 mg of Tris HCl was dissolved in 100 ml of distilled water and the ph was adjusted to Fructose-6-phosphate (1 mm) 3.4 mg of fructose-6-phospahte was dissolved in 10 ml of ATP (1 mm) 5.51 mg of ATP was dissolved in 10 ml of MgCl 2 (2 mm) 4.06 mg of MgCl 2 was dissolved in 10 ml of NADH (0.16 mm) 1.13 mg of NADH was dissolved in 10 ml with Dithiothreitol (2.6 mm) 3.85 mg of dithiothreitol was dissolved in 10 ml of EDTA (1 mm) 3.72 mg of EDTA was dissolved in 10 ml of Ammonium sulphate (5 mm) 6.67 mg of ammonium sulphate was dissolved in 10 ml of Glycogen phosphorylase Citrate buffer (0.1 M, ph 6.0) Solution A: 2.94 g of sodium citrate was dissolved in 100 ml of Solution B: 2.1 g of citric acid was dissolved in 100 ml of 88.5 ml of Solution A and 11.5 ml of Solution B was mixed and the ph was adjusted to Glucose-1-phosphate (0.02 M) 37 mg of glucose-1-phosphate was dissolved in 5 ml of AMP ( M) 8.68 mg of AMP was dissolved in 10 ml of Sodium fluoride (0.075 M) 30 mg of sodium fluoride was dissolved in 10 ml of 158
7 Glycogen (1 %) 50 mg of glycogen was dissolved in 5 ml with Aminonapthosulphonic acid (0.25 %) Solution A: 3 g of sodium bisulphate was dissolved in 20 ml of Solution B: 4 g of sodium sulphite was dissolved in 20 ml of 19.5 ml of Solution A and 0.5 ml of Solution B was mixed and 50 mg of ANSA was dissolved in it Ammonium molybdate (2.5 %) 1.25 g of ammonium molybdate was dissolved in 50 ml of 10 N H 2 SO Bouin s fluid Picric acid (75 ml), formalin (25 ml) and glacial acetic acid (5 ml) was mixed and used as a fixative Glucose-6-Phosphatase Citrate buffer (0.1 M, ph 6.0) Solution A: 2.94 g of sodium citrate was dissolved in 100 ml of Solution B: 2.1 g of citric acid was dissolved in 100 ml of 88.5 ml of Solution A and 11.5 ml of Solution B was mixed and the ph was adjusted to Glucose-6-phosphate (0.02 M) 5.61 mg of glucose-6-phosphate was dissolved in 10 ml of Lysis buffer for immunoblot analyses Lysis buffer for immunoblot analysis g of Tris was dissolved in 100 ml of To this, g of NaCl, 10 ml of glycerol and 1 ml of NP-40 were added and ph was adjusted to 7.4. For every 10 ml of buffer mixture, 50 µl of sodium fluoride (200 mm), 50 µl of sodium orthovanadate (200 mm), and 100 µl of protease inhibitor cocktail [PMSF (1mM), EDTA (1mM), bestatin (150 µm), leupeptin (1 µm) and aprotinin (1 µm)] were added. 159
8 1.17. Immunoblotting Transfer buffer 6.06 g of Tris, 28.8 g of glycine and 2 g of SDS were dissolved in 1 L of distilled. To this, 400 ml of methanol was added and made upto 2 L with distlled water Ponceau S 500 mg of Ponceaus S, 7.5 g of TCA and 7.5 g of sulfosalicylic acid were dissolved in and made up to 25 ml with Phosphate-buffered saline-tris buffer g of Tris was dissolved in To this g of sodium dihydrogen phosphate and g of NaCl were added and ph adjusted to Blocking buffer 5 g of skimmed milk powder was dissolved in 100 ml of PBS-Tris buffer containing 1 % Tween Immunofluorescent staining Phosphate-buffered saline buffer g of sodium dihydrogen phosphate was dissolved in 100 ml of distilled water. To this g of NaCl was added and ph adjusted to % non-immune goat serum 0.1 ml of 10% non-immune goat serum was made upto 10 ml with PBS buffer Mitochondrial membrane potential % trypsin 25 mg of trypsin was made upto 10 ml with DMEM % trypan blue 20 mg trypan blue was made upto 10 ml with PBS Rhodamine 123 Stock solution: 1 mg of Rhodamine 123 was dissolved in 1 ml of distilled water. Working solution: 1 µl of the stock solution was made upto 1000 µl with 160
9 1.20. TUNEL assay % paraformaldehyde solution in PBS 4 ml of paraformaldehyde was dissolved in 100 ml of PBS % H 2 O ml of H 2 O 2 was made up to 100 ml with 161
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