Measurement of Cell Death in Mammalian Cells

Brian S. Cummings1, Lauren P. Wills2, Rick G. Schnellmann2

1 Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, Georgia, 2 Department of Pharmaceutical and Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 12.8
DOI:  10.1002/0471141755.ph1208s56
Online Posting Date:  March, 2012
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Methods for assessing mammalian cell death are presented in this unit. The unit is divided into six sections: (1) a brief overview of cytotoxicity and pathways of cell death, (2) a method to measure cell death using lactate dehydrogenase (LDH) release as a marker of membrane integrity, (3) a flow cytometry method that simultaneously measures two types of cell death, necrosis, and apoptosis, (4) use of fluorescence microscopy and nuclear morphology to assess apoptosis and necrosis, (5) the use of multi‐well plates and high‐content analysis imaging systems to assess nuclear morphology, and (6) a discussion of the use of cytotoxicity assays to determine the mechanisms of cell death. Curr. Protoc. Pharmacol. 56:12.8.1‐12.8.24. © 2012 by John Wiley & Sons, Inc.

Keywords: apoptosis; necrosis; cytotoxicity

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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of Plasma Membrane Integrity and Viability Using LDH Release
  • Basic Protocol 2: Measurement of Necrosis and Apoptosis Using Flow Cytometry
  • Basic Protocol 3: Determination of Nuclear Morphology and Membrane Integrity
  • Alternate Protocol 1: Assessment of Nuclear Morphology and Membrane Integrity Using DAPI and PI
  • Alternate Protocol 2: Assessment of Nuclear Morphology Using Multi‐Well Plates
  • Basic Protocol 4: Measurement of Time‐Dependent Toxicity Using Cell Death Markers
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Measurement of Plasma Membrane Integrity and Viability Using LDH Release

  • Cellular material (∼1 mg protein/ml)
  • Toxicants
  • 2:1 (v/v) n‐butyl phthalate/dioctyl phthalate (store at room temperature for up to 1 month)
  • Cell medium or buffer
  • Liquid nitrogen
  • 16 mM pyruvic acid in LDH‐PO 4 buffer (prepare fresh daily)
  • LDH‐PO 4 buffer: 50 mM K 2HPO 4 and 9 mM KH 2PO 4, pH 7.4 (prepare fresh weekly)
  • 0.3 mM β‐NADH in LDH‐PO 4 buffer (prepare fresh daily)
  • 1.5‐ml microcentrifuge tubes
  • 48‐well transparent plate
  • Fluorometric plate reader with a 360‐nm excitation filter (40‐nm bandwidth) and a 460‐nm emission filter (BMG Laboratories, Molecular Devices, and Bio‐Tek)

Basic Protocol 2: Measurement of Necrosis and Apoptosis Using Flow Cytometry

  • Cells
  • Toxicant (see Critical Parameters)
  • Phosphate‐buffered saline (PBS; Fisher or Sigma)
  • Annexin binding buffer (see recipe)
  • Annexin V–conjugated FITC (annexin V–FITC), typically at 25 µg/ml (i.e., Biovision or R&D Systems)
  • 1 mg/ml propidium iodide (PI) in PBS
  • Orbital shaker
  • Rubber policeman
  • 50‐µm diameter nylon filter sieves
  • 5‐ml snap‐cap polypropylene tubes
  • Flow cytometer with FL‐1 (FITC) and Fl‐2 (PI) channels
NOTE: The amount of annexin V‐FITC and PI needed for flow cytometry varies among cell types. In addition, the amount of FITC conjugated to annexin V can vary from source to source, and lot to lot. Thus, the amount of annexin V‐FITC and PI used to obtain clear and discrete signals may change two‐ to five‐fold for each cell type. Therefore, pilot experiments must be conducted for optimization.

Basic Protocol 3: Determination of Nuclear Morphology and Membrane Integrity

  • Cultured cells on coverslips, in 35‐mm cell culture dishes, or in 96‐well tissue culture plates; or a suspension of cells at a concentration of ∼1 mg protein/ml or 1–2 × 106 cells/ml
  • Phosphate‐buffered saline (PBS)
  • 10% neutral buffered formalin solution (Sigma)
  • 100 µM DAPI in PBS
  • Mounting medium
  • Crystal mount
  • 35‐mm culture dishes
  • Orbital shaker
  • Coverslips
  • Glass slides
  • Fluorometric microscope with 350‐nm excitation and 486‐nm emission filters
  • Centrifuge

Alternate Protocol 1: Assessment of Nuclear Morphology and Membrane Integrity Using DAPI and PI

  • High Content Analysis (HCA) imaging system (e.g., IN Cell Analyzer, GE Healthcare)
  • HCA software package (e.g., Molecular Devices, BD Biosciences, GE Healthcare)
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