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5 protocols using horseradish peroxidase

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Mitochondrial H2O2 Production Assay

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H2O2 production in mitochondria was measured using the Amplex Ultra Red fluorescent reagent (Invitrogen, Carlsbad, CA, USA) according to the protocol described earlier [76 (link)]. The Amplex Red assay is a most specific and sensitive method, with a limit of detection less than 5 pmol of H2O2. The stoichiometry of Amplex Red and H2O2 is 1:1, and, therefore, the assay results are linear over the range of values encountered in tissues and cells [76 (link)]. The excitation wavelength was set to 530 nm and the emission wavelength to 590 nm. The measurements were carried out using a Hitachi F-7000 fluorescence spectrophotometer (Hitachi High Technologies, Tokyo, Japan).
The substrate (10 mM pyruvate), 4 mM phosphate (KH2PO4), 1 U of Amplex Ultra Red reagent, 4 U of horseradish peroxidase (Amresco, Solon, OH, USA), and 0.2 mg of mitochondria were added to 1 mL of isolation buffer (225 mM mannitol, 75 mM sucrose, 5 mM Hepes (pH 7.4), 1 mM EGTA, 2 mg/mL fatty acids free BSA. The H2O2 concentration was measured as the fluorescence intensity of resorufin formed during the reaction upon oxidation of Amplex Ultra Red. Production changes were recorded after the addition of 0.2 mg cisplatin, 1 μM methylene blue, and 1 μM azure B.
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2

Monoclonal Antibody Production and Purification

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To produce mAbs, 2 × 106 selected hybridoma cells were administered intraperitoneally into 20-week-old BALB/c mice. Selected mAbs were purified by ammonium sulfate precipitation from ascitic fluids and then purified using the protein A chromatography (GE Healthcare, USA). The purity and size of the purified IgG antibodies were examined by SDS-PAGE and western blot analyses. Purified mAbs were resolved by 12.5% SDS-PAGE under reducing conditions and transferred onto nitrocellulose membrane (Bio-Rad). After blocking by 5% dry skim milk, the membrane was incubated with anti-mouse IgG alkaline phosphatase-conjugated goat IgG (Sigma Aldrich, USA). Immune complexes were visualized by a mixture of nitro blue tetrazolium (NBT, Amresco) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP, Roche) for 20 min.
Selected mAbs were conjugated with horse-radish peroxidase (Amresco, USA) using optimized NaIO4 method as described previously [36 (link)].
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Phospholipase D Activity Assay

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Hydroxychloroquine, PLD assay reagent amplex red 10-Acetyl-3,7-dihydroxyphenoxazine, and 2-dioctanoyl-sn-glycero-3-phosphocholine (C8-PC) were purchase from Cayman Chemical and tetracaine, methylbetacyclodextrin (MβCD), and cabbage PLD were purchased from Sigma–Aldrich. Horseradish peroxidase and choline oxidase were purchased from VWR.
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4

Phospholipase D2 Activity Assay

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Hydroxychloroquine was purchase from Cayman Chemical and tetracaine was purchased from Sigma-Aldrich. Purified PLD2 from cabbage was purchased from Sigma-Aldrich respectively. PLD assay reagent amplex red 10-Acetyl-3,7-dihydroxyphenoxazine and 2-dioctanoyl-sn-glycero-3-phosphocholine (C8-PC) were purchased from Cayman Chemical. Horseradish peroxidase and choline oxidase were purchased from VWR. Methylbetacyclodextrin (MβCD) was purchased from Sigma-Aldrich.
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5

Lipid Substrate Assay for Phospholipase D

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Bacterial (Streptomyces chromofuscus), peanut, and cabbage PLD was purchased from Enzo and sigma-aldrich respectively. Amplex Red 10-Acetyl-3,7-dihydroxyphenoxazine (Cayman chemical). Horseradish peroxidase (VWR), choline oxidase (VWR). All lipids including 1,2-dioctanoyl-sn-glycero-3-phosphocholine (C8-PC), 1-palmitoyl-2-(dipyrrometheneboron difluoride) undecanoyl-sn-glycero-3-phosphocholine (TopFluorPC), 1-(dipyrrometheneboron difluoride) undecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (TopFluorLPC), 1 -palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC or 18:1 PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol (POPEtOH), and 1,2-dioleoyl-sn-glycero-3-phosphoethanol (DOPEtOH) were purchased from Avanti Polar Lipids (Alabaster, AL) in chloroform at stock concentrations of 10 or 25 mg/mL except C8-PC and TopFluor lipids which were purchased as powder. Carbonyl cyanide m-chlorophenyl hydrazine (CCCP) was from Tocris Bioscience and made at a stock concentration of 1mM in ethanol. Valinomycin was also purchased from Tocris Bioscience and made at a stock concentration of 200μM in ethanol. The detergent n-dodecyl-β-D-maltoside (DDM) was purchased from Anatrace (D310S) and made at 200mM. ACMA (9-amino-6-chloro-2-methoxyacridine), was purchased from Life Technologies and made at a stock concentration of 2mM in ethanol.
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