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. 2006 Jun;1(2):125–131. doi: 10.1007/BF02829954

Techniques for quantifying effects of dietary antioxidants on transcription factor translocation and nitric oxide production in cultured cells

B A Ewins 1,✉, M Vassiliadou 1, A M Minihane 1, G H Rimbach 2, P D Weinberg 3,✉
PMCID: PMC3454682  PMID: 18850206

Abstract

Dietary antioxidants can affect cellular processes relevant to chronic inflammatory diseases such as atherosclerosis. We have used non-standard techniques to quantify effects of the antioxidant soy isoflavones genistein and daidzein on translocation of Nuclear Factor-KB (NF-KB) and nitric oxide (NO) production, which are important in these diseases. Translocation was quantified using confocal immunofluoresecence microscopy and ratiometric image analysis. NO was quantified by an electrochemical method after reduction of its oxidation products in cell culture supernatants. Activation of the RAW 264.7 murine monocytel macrophage cell line increased the ratio of nuclear to cytoplasmic immunostaining for NF-κB. The increase was exacerbated by pre-treatment with genistein or daidzein. To show that decreases could also be detected, pre-treatment with the pine bark extract Pycnogenol® was examined, and found to reduce translocation. NO production was also increased by activation, but was reduced by pre-treatment with genistein or daidzein. In the EA.hy926 human endothelial cell line, constitutive production was detectable and was increased by thrombin. The confocal and electrochemical methods gave data that agreed with results obtained using the established electromobility shift and Griess assays, but were more sensitive, more convenient, gave more detailed information and avoided the use of radioisotopes.

Keywords: Confocal Microscopy, Daidzein, Endothelium, Genistein, Macrophage, Microelectrode, Nuclear Factor Kappa-B

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