Dataset Information

Flow Cytometric Analysis of Efflux by Dye Accumulation.

ABSTRACT: Gram-negative infections are increasingly difficult to treat because of their impermeable outer membranes (OM) and efflux pumps which maintain a low intracellular accumulation of antibiotics within cells. Historically, measurement of accumulation of drugs or dyes within Gram-negative cells has concentrated on analyzing whole bacterial populations. Here, we have developed a method to measure the intracellular accumulation of ethidium bromide, a fluorescent DNA intercalating dye, in single cells using flow cytometry. Bacterial cells were stained with SYTOTM 84 to easily separate cells from background cell debris. Ethidium bromide fluorescence was then measured within the SYTOTM 84 positive population to measure accumulation. In S. Typhimurium SL1344, ethidium bromide accumulation was low, however, in a number of efflux mutants, accumulation of ethidium bromide increased more than twofold, comparable to previous whole population analysis of accumulation. We demonstrate simultaneous measurement of ethidium bromide accumulation and GFP allowing quantification of gene expression or other facets of phenotype in single cells. In addition, we show here that this assay can be adapted for use with efflux inhibitors, with both Gram-negative and Gram-positive bacteria, and with other fluorescent substrates with different fluorescence spectra.

SUBMITTER: Whittle EE

PROVIDER: S-EPMC6787898 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

Flow Cytometric Analysis of Efflux by Dye Accumulation.

Whittle Emily E EE Legood Simon W SW Alav Ilyas I Dulyayangkul Punyawee P Overton Tim W TW Blair Jessica M A JMA

Frontiers in microbiology 20191004

Gram-negative infections are increasingly difficult to treat because of their impermeable outer membranes (OM) and efflux pumps which maintain a low intracellular accumulation of antibiotics within cells. Historically, measurement of accumulation of drugs or dyes within Gram-negative cells has concentrated on analyzing whole bacterial populations. Here, we have developed a method to measure the intracellular accumulation of ethidium bromide, a fluorescent DNA intercalating dye, in single cells u ...[more]

PMID: 31636625

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Project description:BACKGROUND: A useful application of flow cytometry is the investigation of cell receptor-ligand interactions. However such analyses are often compromised due to problems interpreting changes in ligand binding where the receptor expression is not constant. Commonly, problems are encountered due to cell treatments resulting in altered receptor expression levels, or when cell lines expressing a transfected receptor with variable expression are being compared. To overcome this limitation we have developed a Microsoft Excel spreadsheet that aims to automatically and effectively simplify flow cytometric data and perform statistical tests in order to provide a clearer graphical representation of results. RESULTS: To demonstrate the use and advantages of this new spreadsheet method we have investigated the binding of the transmembrane adhesion receptor CD44 to its ligand hyaluronan. In the first example, phorbol ester treatment of cells results in both increased CD44 expression and increased hyaluronan binding. By applying the spreadsheet method we effectively demonstrate that this increased ligand binding results from receptor activation. In the second example we have compared AKR1 cells transfected either with wild type CD44 (WT CD44) or a mutant with a truncated cytoplasmic domain (CD44-T). These two populations do not have equivalent receptor expression levels but by using the spreadsheet method hyaluronan binding could be compared without the need to generate single cell clones or FACS sorting the cells for matching CD44 expression. By this method it was demonstrated that hyaluronan binding requires a threshold expression of CD44 and that this threshold is higher for CD44-T. However, at high CD44-T expression, binding was equivalent to WT CD44 indicating that the cytoplasmic domain has a role in presenting the receptor at the cell surface in a form required for efficient hyaluronan binding rather than modulating receptor activity. CONCLUSION: Using the attached spreadsheets and instructions, a simple post-acquisition method for analysing bivariate flow cytometry data is provided. This method constitutes a straightforward improvement over the standard graphical output of flow cytometric data and has the significant advantage that ligand binding can be compared between cell populations irrespective of receptor expression levels.

Dataset Information

Flow Cytometric Analysis of Efflux by Dye Accumulation.

Publications

Flow Cytometric Analysis of Efflux by Dye Accumulation.

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