In Vitro Methods for Detecting Cytotoxicity

Marion Ehrich1, Lioudmilla Sharova1

1 Virginia‐Maryland Regional College of Veterinary Medicine, Blacksburg, Virginia
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.6
DOI:  10.1002/0471140856.tx0206s03
Online Posting Date:  May, 2001
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Abstract

Toxicant‐induced damage to cells (cytotoxicity) can depress cell growth, compromise intracellular metabolic processes, and/or cause loss of cell viability. Methods that indicate these cytotoxic changes following toxicant exposures are provided, including [3H]thymidine uptake to measure cell growth, MTT dye conversion to detect changes in metabolic activity, and trypan blue uptake to indicate loss of cell viability.

     
 
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Table of Contents

  • Basic Protocol 1: Trypan Blue Uptake to Assess Viability
  • Basic Protocol 2: Lactate Dehydrogenase (LDH) Leakage to Assess Viability
  • Alternate Protocol 1: In Situ LDH Assay to Assess Viability
  • Alternate Protocol 2: Phenylhydrazine Colorimetric LDH Assay to Assess Viability
  • Alternate Protocol 3: Int Colorimetric LDH Assay to Assess Viability
  • Alternate Protocol 4: Kinetic Int Colorimetric LDH Assay to Assess Viability
  • Basic Protocol 3: Neutral Red Dye Retention to Assess Viability
  • Alternate Protocol 5: Neutral Red Endpoint Assay to Assess Viability
  • Basic Protocol 4: Propidium Iodide Attachment to Double‐Stranded Nucleic Acids to Assess Viability
  • Alternate Protocol 6: Using Fluorescent Markers to Assess Viability
  • Basic Protocol 5: Assays to Assess Cell Growth
  • Basic Protocol 6: [3H]Thymidine Uptake to Assess Cell Growth
  • Basic Protocol 7: Cell Cycle Analysis with Propidium Iodide to Assess Cell Growth
  • Basic Protocol 8: MTT Dye Conversion to Assess Metabolic Capability
  • Basic Protocol 9: Alamar Blue Reduction to Assess Metabolic Capability
  • Basic Protocol 10: Measuring Fluorescent Dye Loss to Assess Metabolic Capability
  • Alternate Protocol 7: USING [3H]2‐Deoxy‐D‐Glucose to Assess Metabolic Capability
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Trypan Blue Uptake to Assess Viability

  Materials
  • Cells treated with toxicant, vehicle, or 10% (v/v) Triton X‐100 in suspension
  • Phosphate‐buffered saline (PBS; see recipe), pH 7.2 to 7.4
  • 0.1% (w/v) trypan blue (see recipe)
  • Hemocytometer
  • Phase‐contrast inverted microscope
  • Hand counter

Basic Protocol 2: Lactate Dehydrogenase (LDH) Leakage to Assess Viability

  Materials
  • Cells
  • Test compounds: toxicant, vehicle, and Triton X‐100
  • LDH Reagent Kit (DG 1340K, Sigma) containing:
    • NADH, 0.194 mmol/liter in phosphate buffer
    • Phosphate buffer, 54 mmol/liter, pH 7.5
    • Pyruvate, 6.48 mmol/liter in phosphate buffer
  • 96‐well microtiter plates, for cell culture
  • Microtiter plate spectrophotometer with wavelength at 340 nm

Alternate Protocol 1: In Situ LDH Assay to Assess Viability

  Materials
  • 1 to 2 × 105 cells/ml treated with toxicant, vehicle, or 10% (v/v) Triton X‐100
  • 36 mg/ml lactate in 10 mM Tris⋅Cl, pH 8.5 ( appendix 2B)
  • INT dye (see recipe)
  • NAD+ solution (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe), pH 7.2 to 7.4
  • 16 mg/ml oxymate (oxamic acid, sodium salt) in recipePBS
  • Microtiter plate reader at 490 nm

Alternate Protocol 2: Phenylhydrazine Colorimetric LDH Assay to Assess Viability

  Materials
  • 105 cells/ml cell suspension
  • Test compound: toxicant, vehicle, or positive control (1% w/v SDS or 1% w/v saponin)
  • Phosphate buffered saline (PBS; see recipe)
  • 1% (w/v) SDS
  • Neutral red solution (see recipe)
  • Dye extractor solution (see recipe)
  • Flat‐bottomed, 96‐well microtiter plates, cell culture coated
  • Blotter paper (e.g., absorbent paper towels or filter paper)
  • Plate mixer
  • Microtiter plate reader, 550 nm

Alternate Protocol 3: Int Colorimetric LDH Assay to Assess Viability

  Materials
  • 0.5 × 105 cells/ml
  • Test compounds: toxicant, vehicle, and positive control
  • Phosphate buffered saline (PBS; see recipe)
  • 376 µM digitonin solution
  • 30 µM propidium iodide dye (Molecular Probes)
  • Flat bottomed, 96‐well microtiter plates, for cell culture
  • Fluorescence microtiter plate reader

Alternate Protocol 4: Kinetic Int Colorimetric LDH Assay to Assess Viability

  Materials
  • Cells (1 to 2 × 105 cells/well) or 150 µl of 1 × 106 cells/ml, treated with toxicant, vehicle, or control in microtiter plates
  • Phosphate‐buffered saline (PBS; see recipe), 37°C
  • Fixative: 1% (v/v) glacial acetic acid/50% (v/v) ethanol/49% (v/v) water
  • Kenacid blue dye (see recipe)
  • Washing solution: 10% (v/v) ethanol/5% (v/v) glacial acetic acid/85% (v/v) water
  • Microtiter plate reader, 577 nm

Basic Protocol 3: Neutral Red Dye Retention to Assess Viability

  Materials
  • 1 to 15 × 105 cells/ml
  • Serum‐free chemically‐defined medium (e.g., RPMI 1640 or DMEM), 37°C and ice‐cold
  • Test compounds(s): toxicant and vehicle
  • 20× [3H]thymidine solution (see recipe)
  • 10% (w/v) trichloroacetic acid (TCA) solution, ice‐cold
  • 0.3 N sodium hydroxide
  • Scintillation fluid
  • 24‐well plates, coated for cell culture
  • Liquid scintillation counter
  • Radioactive waste disposal system
CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area following the guidelines provided by your local radiation safety offices (also see appendix 1A).CAUTION: Trichloroacetic acid is very caustic. Handle it with care.NOTE: All incubations should be performed at 37°C using an incubator or water bath.

Alternate Protocol 5: Neutral Red Endpoint Assay to Assess Viability

  Materials
  • Toxicant‐treated and control cells in suspension
  • Standard azide buffer (see recipe)
  • Vindelov's PI (see recipe)
  • 12 × 75–mm clear polystyrene tubes
  • 30‐µm mesh (optional)
  • Flow cytometer

Basic Protocol 4: Propidium Iodide Attachment to Double‐Stranded Nucleic Acids to Assess Viability

  Materials
  • 5 × 104 cells/ml cell suspension
  • Test compound: toxicant or vehicle
  • Culture medium
  • 5 mg/ml MTT in recipePBS (store 2 to 3 weeks at 4°C or longer at −20°C)
  • Dimethyl sulfoxide (DMSO), photometric grade
  • Sorensen's buffer (see recipe)
  • 96‐well microtiter plates, for cell culture
  • Aspiration system for removal of medium from cells
  • Micropipettor (20 to 200 µl) with sterile micropipettor tips
  • Microtiter plate reader

Alternate Protocol 6: Using Fluorescent Markers to Assess Viability

  Materials
  • Cells in microtiter plates
  • Test compound: toxicant or vehicle
  • Alamar blue assay kit (Biosource International)
  • Microtiter plate reader
  • Additional reagents and equipment for counting cells and assessing viability using trypan blue dye ( protocol 1)

Basic Protocol 5: Assays to Assess Cell Growth

  Materials
  • Cells
  • Test compound: toxicant or vehicle
  • 50 µg/ml CFDA‐AM in recipePBS (prepared fresh)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Fluorescence microtiter plate reader

Basic Protocol 6: [3H]Thymidine Uptake to Assess Cell Growth

  Materials
  • 106 cells grown in 60‐mm culture dishes
  • Test compound: toxicant or vehicle
  • Phosphate‐buffered saline (PBS; see recipe)
  • 0.55 µCi/ml [3H]2‐deoxy‐D‐glucose in recipePBS
  • 1 mg/ml glucose in recipePBS
  • 1 M NaOH
  • 1 M HCl
  • Scintillation fluid
CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area following the guidelines provided by your local radiation safety offices (also see appendix 1A).
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Figures

Videos

Literature Cited

Literature Cited
   Abdulla, E.M. and Campbell, I.C. 1993. Use of neurite outgrowth as an in vitro method of assessing neurotoxicity. In Markers of Neuronal Injury and Degeneration (J.N. Johansson, ed.) pp. 276‐279. Ann. N.Y. Acad. Sci., 679.
   Ahmed, A., Gogal, R.M., and Walsh, Y.E. 1994. A new rapid and simple non‐radioactive assay to monitor and determine the proliferation of lymphocytes: An alternative to [3H]thymidine incorporation assay. J. Immunol. Methods 170:211‐224.
   Babich, H. and Borenfreund, E. 1992. Neutral red assay for toxicology in vitro. In In Vitro Methods of Toxicology (R.R. Watson, ed.) pp. 237‐251. CRC Press, Boca Raton, Fla.
   Barile, F.A. 1994. Introduction to In Vitro Cytotoxicology Mechanisms and Methods. CRC Press, Boca Raton, Fla.
   Forsby, A., Pilli, F., Bianchi, V., and Walum, E. 1995. Determination of critical cellular neurotoxic concentrations in human neuroblastoma (SH‐SY5Y) cell cultures. Alternatives to Lab. Animals 23:800‐811.
   Haugland, R.P. 1996. Handbook of Fluorescent Probes and Research Chemicals, 6th ed. Molecular Probes, Eugene, OR.
   Krishan, A. 1975. Rapid flow cytometric analysis of mammalian cell cycle by propidium iodine staining. J. Cell Biol. 66:188‐193.
   Mosman, T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55‐63.
   Nieminen, A.L., Gores, G.J., Bond, J.M., Imberti, R., Herman, B., and LeMaster, J.J. 1992. A novel cytotoxicity screening assay using a multi‐well fluorescence scanner. Toxicol. Appl. Pharmacol. 115:147‐155.
   O'Hare, S. and Atterwill, C.K. 1995. Methods in Molecular Biology 43: In Vitro Toxicity Testing Procedures. Human Press, Totowa, N.J.
   Rowles, T.R., Song, X., and Ehrich, M. 1995. Identification of endpoints affected by exposure of human neuroblastoma cells to neurotoxicants at concentrations below those that affect cell viability. In Vitro Toxicol. 8:3‐13.
   Song, X., Perkins, S., Jortner, B.S., and Ehrich, M. 1997. Cytotoxic effects of MPTP on SH‐SY5Y human neuroblastoma cells. Neurotoxicology 18:341‐354.
   Veronesi, B. and Ehrich, M. 1993. Differential cytotoxic sensitivity in mouse and human cell lines exposed to organophosphate insecticides. Toxicol. Appl. Pharmacol. 120:240‐246.
Key References
   Alley, M.C., Scudiero, D.A., Monks, A., Hursey, M.L., Czerwinski, M.J., Fine, D.L., Abbot, B.J., Mayo, J.G., Shoemaker, R.H., and Boyd, M.R. 1988. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res. 48:589‐601.
  This describes a microassay for MTT ().
   Application notes for the Cytofluor Fluorescent Plate Reader, Millipore Corporation, Marlborough, MA.
  These notes discuss use of multiple probes for multiple endpoints in a single incubate of cells, providing excitation and emission data, that can be used when fluorescent dye loss () is used to indicate cytotoxicity.
   Borenfreund, E. and Puerner, J.A. 1985. A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR‐90). J. Tissue Cult. Methods 9:7‐9.
  This is an early paper from a laboratory with recognized expertise with the neural red assay ( and ).
   Nieminen et al., 1992. See above.
  Optimization of the procedure for measuring cell viability by propidium iodide uptake () is described in this reference.
   Technical Information, Alamar Blue Assay, Biosource International, Camarillo, CA.
  Details about the alamar blue assay system () and how to use it are provided.
   Technical Information included with LDH Assay Kits, Sigma Chemical, St. Louis, MO.
  These provide specific details for use of their kits when LDH leakage ( and Alternate Protocols 1 to 4) is used as the indicator of loss of cell viability.
   Technical Information on Trypan Blue, Sigma Chemical, St. Louis, MO.
  This provides a detailed protocol, including a diagram of a hemocytometer, for the trypan blue assay ().
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