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Viability Assays for Cells in Culture
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Viability Assays for Cells in Culture

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Streamlined duplex live-dead microplate assay for cultured cells.

Bruce A Pfeffer1, Steven J Fliesler2

  • 1Department of Ophthalmology, University at Buffalo-State University of New York (SUNY), Buffalo, NY, USA; SUNY Eye Institute, Buffalo, NY, USA.

Experimental Eye Research
|June 3, 2017
PubMed
Summary

This study presents a novel duplex fluorescence assay for cell viability testing in multi-well plates. The assay uses readily available reagents and measures both live and dead cells directly in the culture plate, enhancing experimental efficiency.

Keywords:
Calcein AMCell viability assayFluorescence assayPlate readerRetinal cell lineSytox Orange

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Area of Science:

  • Cell Biology
  • Biochemistry
  • Assay Development

Background:

  • Assessing cell viability is crucial in biological research, particularly when evaluating the effects of experimental treatments.
  • Existing cell viability assays often require multiple steps, including cell transfer or medium exchange, increasing the risk of error and reducing efficiency.
  • The presence of efflux pumps in live cells can affect assay accuracy by removing viability indicators.

Purpose of the Study:

  • To develop a streamlined, duplex fluorescence assay for assessing cell viability in multi-well plates.
  • To enable direct measurement of live and dead cells within the original culture plate without cell manipulation.
  • To improve assay dynamic range by inhibiting cellular efflux mechanisms.

Main Methods:

  • A two-color fluorescence assay was developed using calcein acetoxymethyl ester (CaAM) for live cells and SYTOX® Orange (SO) for dead cells.
  • CaAM measures esterase activity in live cells, producing a green fluorescent signal.
  • SO penetrates dead cells with compromised membranes, binding to DNA and emitting an orange fluorescent signal.
  • An inhibitor of plasma membrane multidrug transport proteins (e.g., ABC transporters) was included during CaAM incubation to enhance assay sensitivity.

Main Results:

  • The assay successfully distinguishes between live and dead cells based on distinct fluorescence signals (∼530 nm for live, ∼590 nm for dead).
  • The duplex assay can be performed in the original culture plate, eliminating the need for cell transfer or medium exchange.
  • Inclusion of an efflux pump inhibitor improved the assay's dynamic range.
  • The assay demonstrated its utility in evaluating the effects of cytotoxic compounds with different mechanisms of action.

Conclusions:

  • A robust and efficient duplex fluorescence assay for cell viability assessment has been established.
  • This method simplifies cell viability testing, reducing potential errors and saving time.
  • The assay is adaptable for various applications, including drug screening and toxicity studies, and allows for direct correlation with cell morphology.