Determination of Metabolic Viability and Cell Mass Using a Tandem Resazurin/Sulforhodamine B Assay

Filomena S.G. Silva1, Irina G. Starostina1, Vilena V. Ivanova2, Albert A. Rizvanov2, Paulo J. Oliveira3, Susana P. Pereira4

1 These authors should be considered co–first authors, 2 Kazan Federal University, Institute of Fundamental Medicine and Biology, Kazan, Russia, 3 Center for Neuroscience and Cell Biology, University of Coimbra, Cantanhede, Portugal, 4 Department of Life Sciences, School of Sciences and Technology, University of Coimbra, Coimbra, Portugal
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.24
DOI:  10.1002/cptx.1
Online Posting Date:  May, 2016
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The identification of rapid, reliable, and highly reproducible biological assays that can be standardized and routinely used in preclinical tests constitutes a promising approach to reducing drug discovery costs and time. This unit details a tandem, rapid, and reliable cell viability method for preliminary screening of chemical compounds. This assay measures metabolic activity and cell mass in the same cell sample using a dual resazurin/sulforhodamine B assay, eliminating the variation associated with cell seeding and excessive manipulations in assays that test different cell samples across plates. The procedure also reduces the amount of cells, test compound, and reagents required, as well as the time expended in conventional tests, thus resulting in a more confident prediction of toxic thresholds for the tested compounds. © 2016 by John Wiley & Sons, Inc.

Keywords: high throughput; human skin fibroblasts; preclinical tests; toxicity evaluation

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Table of Contents

  • Introduction
  • Basic Protocol 1: Cell Culture of Fibroblasts
  • Basic Protocol 2: Resazurin Assay for Cell Viability Assessment
  • Basic Protocol 3: Sulforhodamine B Colorimetric Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Cell Culture of Fibroblasts

  • BJ skin fibroblasts cell line (ATCC, cat. no CRL‐2522)
  • Phosphate buffered saline (PBS; see recipe)
  • 0.05% trypsin‐EDTA (Life Technologies, cat. no 25300‐062)
  • Growth medium (see recipe)
  • 100‐cm2 cell culture dishes
  • Cell incubator, at 37°C with 5% CO 2 atmosphere
  • Inverted microscope
  • 15‐ml disposable conical polypropylene centrifuge tubes (e.g., Corning Falcon)

Basic Protocol 2: Resazurin Assay for Cell Viability Assessment

  • Cells seeded in 48‐well plates (see protocol 1)
  • Resazurin solution (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Growth medium (see recipe)
  • Microplate reader
  • Computer running MS Excel and GraphPad Prism or SPSS (IBM)

Basic Protocol 3: Sulforhodamine B Colorimetric Assay

  • Cells seeded in 48‐well plates, after performing resazurin assay for all time points (see protocol 2)
  • Phosphate‐buffered saline (PBS)
  • 1% (v/v) acetic acid in methanol (see recipe)
  • SRB solution (see recipe)
  • 1% (v/v) acetic acid in MilliQ‐purified H 2O
  • 10 mM Tris‐NaOH, pH 10 (see recipe)
  • 96‐well plates suitable for spectrometry
  • Orbital plate shaker
  • Microplate reader
  • Computer running MS Excel and GraphPad Prism or SPSS (IBM)
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Literature Cited

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