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2.11.1 Measurement of reactive oxygen species

2.11.1.1 Using 2’-7’dichlorofluorescin (DCFH)

To measure reactive oxygen species (ROS) the probe, 5-(and-6)-chloromethyl-2’,7’- dicholorodihydrofluorescien diacetate, acetyl ester (CM-H2DCFDA) (Molecular probes, Invitrogen) was used. CM-H2DCFDA is a cell permeable indicator for reactive oxygen species which is retained inside the cells after cleavage of acetate moiety by esterases. This compound then reacts with intracellular ROS changing its fluorescent properties which can be quantified. Shortly before performing the experiment, the CM-H2DCFDA is reconstituted in 100% ethanol to make a stock concentration of 20 μM. Cells were then washed twice in sPBS (37°C), and incubated with the ROS indicator in phenol free media for 30 minutes. Due to the dye’s susceptibility to photo-oxidation, care was taken to avoid light exposure, and hence low light conditions were used where possible. After the 30 minute incubation, the media was removed and the cells washed twice in PBS. To measure ROS using flow cytometry, cells were collected in 1 × trypsin-EDTA, 37°C for 5 min and then neutralised using an equal volume of TNS. Cells were then pelleted by centrifugation (800 × g, 10 min), the supernatant was removed and the cell pellet was homogenised in approximately 1 ml PBS. Cell acquisition and analysis was carried out in the CyAN flow cytometer, (Dako Becton-Dickinson). A minimum of 10,000 cell events was counted for each sample and non-staining samples were used for control. To measure ROS using a fluorometer cells were seeded in a black-edged 96 well plate at 10,000 cells per well. Cells were seeded in phenol free media, in quadruple wells and left overnight to adhere. The following day cells washed twice in sPBS (37°C), and incubated with the ROS indicator in phenol free media for 30 minutes (covered with foil to reduce excessive light exposure). The probe was then removed and the cells washed twice in sPBS (37°C) before adding media containing defined treatments. Cells without the probe were used as negative controls to correct for background fluorescence. The plate was then placed in a Victor 2, 1420 Multi label counter, in a pre-warmed reading chamber (37˚C). Excitation and emission was measured at 488 nm and 520 nm, respectively (Wallac Victor 2, 1420 Multi label counter, Perkin Elmer). Sequential readings were taken every 10 minutes for up to 1 hour. At the end of the experiment protein concentration (see section 2.3) was determined to ensure equal loading of cells.

2.11.1.2 Using HyPer

HyPer consists of yellow fluorescent protein (YFP) inserted into the regulatory domain of the prokaryotic H2O2-sensing protein, OxyR (Belousov et al., 2006). Cells were transfected with the plasmid carrying HyPer using Lipofectamine transfection reagent following the protocol previously described in section 2.9.1. HyPer-transfected cells exhibiting yellow florescent protein (YFP) positive staining were then selected using fluorescence-activated cell sorting (FACS), as the plasmid has no antibiotic selection in mammalian cells. FACS analysis was performed by Scientific Support Services (Wolfson Institute for Biomedical Research, UCL) using a MoFlow high-speed cell sorter. HyPer was kindly provided by Dr A Galkin, Wolfson Institute for Biomedical Research, UCL, London, UK. Following sorting, HyPer transfected cells were incubated with defined treatments and cell acquisition and analysis was carried out in the CyAN flow cytometer, (Dako Becton-Dickinson). A minimum of 10,000 cell events was counted for each sample and non-staining samples were used for control.

2.11.2 Nitrite quantification

The amount of nitric oxide (NO) was estimated by measuring nitrite (NO2-) levels in the extracellular medium using the Griess reagent kit (Molecular Probes, Leiden, Netherlands). Total nitrite was quantified by the conversion of sulfanilic acid to a diazonium salt by the reaction with nitrite in acid solution. The diazonium salt is then coupled to N-(1-naphthyl)ethylenediamine, forming an azo dye that brings about a colourimetric change which is spectorophotometrically detected. Nitrite concentrations were determined based on a standard curve produced from a series of dilutions of known concentrations of NO2-. 50 µl of sample or standard were mixed with 50 µl Griess reagent. Assays were performed in triplicate in a 96-well plate, and incubated for 10 minutes in the dark at room temperature. Absorbance was measured at 540 nm and 620 nm (Molecular Devices, SpectraMax Plus).

2.11.3 Lactate quantification

Lactate concentration was determined using a lactate oxidase-based assay (Trinity Biotech Ltd). Total lactate was quantified by the conversion of lactic acid to pyruvate and hydrogen peroxide (H2O2) by lactate oxidase. In the presence of the H2O2 formed,

a coloured dye with an absorption maximum at 540 nm. The increase in absorbance at 540 nm is directly proportional to the lactate concentration in the sample. 5 µl sample was added to a 96 well plate in triplicate, followed by 100 µl lactate reagent. The plate is then shaken briefly on a shaker table and then incubated in the dark for 10 minutes. Absorbance was measured at 540 nm (Molecular Devices, SpectraMax Plus).

2.11.4 Measurement of total glutathione

Total cellular glutathione (GSH) was measured in HEK 293T cells using a colorimetric assay GSH-400 kit (Oxis Research, Portland, USA). Glutathione was measured via a two-step chemical reaction which first involves the formation of thioethers between 4- chloro-1 –methyl-7-trifluromethyl-quinolinium methylsulfate (R1) and all mercaptans (RSH) present in the samples. The second step involves a β-elimation reaction which takes places under alkaline conditions, allowing for the transformation of the thioethers obtained from GSH into a chromophoric thione which has a maximal absorbance wavelength at 400 nm. Samples were collected in approximately 2 ml 1 × trypsin- EDTA, 37°C for 5 min. Cells were then pelleted by centrifugation (800 × g, 10 min), the supernatant was removed and the cell pellet was homogenised in 500 µl Metaphosphoric acid (MPA). Cells were then centrifuged at 3000 × g for 10 minutes, and the clear upper aqueous layer was collected. Then both samples and standards were prepared in a 900 µl final volume. 50 µl solution R1 was added, mixed thoroughly, followed by the addition of 50 µl solution R2, and mixed thoroughly again. The samples were then incubated at 25°C for 10 minutes in the dark. Absorbance was measured at 400 nm (Varian, Cary 4000 UV-Vis Spectrophotometer).