Development of a fluorometric microplate antiadhesion assay using uropathogenic Escherichia coli and human uroepithelial cells - PubMed (original) (raw)

. 2014 May 23;77(5):1102-10.

doi: 10.1021/np400781y. Epub 2014 Apr 22.

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Development of a fluorometric microplate antiadhesion assay using uropathogenic Escherichia coli and human uroepithelial cells

Lindsey L Kimble et al. J Nat Prod. 2014.

Abstract

A fluorometric microplate assay has been developed to determine Escherichia (E.) coli adhesion to uroepithelial cells (UEC). P-fimbriated E. coli were labeled with BacLight Green and preincubated 30 min with human urine or standard. Fluorescent-E. coli were added to UEC in mircoplates at a 400:1 ratio, incubated 1 h, and washed, and the fluorescence intensity was measured. Specific labeling and adherence were confirmed by flow cytometry. A myricetin (1) standard curve (0-30 μg/mL) was developed; the lower limit of detection was 0.1 μg/mL, and half-maximal inhibitory concentration was 0.88 μg/mL (intra- and interassay coefficients of variance were <10% and <15%, respectively). Vaccinium macrocarpon (cranberry) extracts, quercetin (2), and procyanidins B1 (3), B2 (4), and C1 (5) showed similar inhibition. Antiadhesion activity of urine samples from subjects (n = 12) consuming placebo or V. macrocarpon beverage determined using this assay was positively correlated (R(2) = 0.78; p < 0.01) with a radiolabeled-E. coli assay.

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