Assessment of Cell Viability in Primary Neuronal Cultures

Mandar A. Aras1, Karen A. Hartnett1, Elias Aizenman1

1 Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 7.18
DOI:  10.1002/0471142301.ns0718s44
Online Posting Date:  July, 2008
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit contains five protocols for assaying cell viability in vitro using primary neuronal cultures, including a novel method for use with transfected neurons. Three of the assays are based on the principle that cell death cascades alter membrane permeability. The lactate dehydrogenase (LDH) release assay measures the amount of the cytoplasmic enzyme released into the bathing medium, while the trypan blue and propidium iodide assays measure the ability of cells to exclude dye from their cytoplasm. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay measures the mitochondrial activity of viable cells by quantifying the conversion of the tetrazolium salt to its formazan product. Finally, the fifth assay details the measurement of luciferase expression as an indication of neuronal viability within a relatively small population of transfected neurons. Curr. Protoc. Neurosci. 44:7.18.1‐7.18.15. © 2008 by John Wiley & Sons, Inc.

Keywords: neuronal viability; lactate dehydrogenase; MTT; trypan blue; propidium iodide; firefly luciferase

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Cell Viability Assay by Measurement of Lactate Dehydrogenase (LDH) Release
  • Alternate Protocol 1: Cell Viability Assay by Measurement of Total Cytoplasmic LDH
  • Basic Protocol 2: Cell Viability Assay by Measurement of Mitochondrial Activity (MTT Reduction)
  • Basic Protocol 3: Cell Viability Assay by Colloidal Dye (Trypan Blue) Exclusion
  • Basic Protocol 4: Cell Viability Assay Using Propidium Iodide
  • Support Protocol 1: Preservation of Stained Cells
  • Basic Protocol 5: Transfected Cell Viability Assay by Firefly Luciferase Activity
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Cell Viability Assay by Measurement of Lactate Dehydrogenase (LDH) Release

  Materials
  • Cells in 24‐well culture plate (500 µl bathing medium/well)
  • Minimal essential media (MEM without phenol red; Invitrogen)
  • TOX7 kit (LDH‐based in vitro toxicology assay kit, Sigma cat. no. TOX7‐1KT) containing:
    • LDH substrate
    • LDH enzyme prep
    • LDH dye solution
    • LDH lysis solution
  • 96‐well flat‐bottom, clear microtiter plate
  • Repeater pipet
  • Microplate spectrophotometer (e.g., Wallac 1420 Victor2 multilabel counter, PerkinElmer)

Alternate Protocol 1: Cell Viability Assay by Measurement of Total Cytoplasmic LDH

  • LDH lysis solution (Sigma cat. no. L2152)

Basic Protocol 2: Cell Viability Assay by Measurement of Mitochondrial Activity (MTT Reduction)

  Materials
  • Cells in 24‐well plate (500 µl bathing medium/well)
  • 5 mg/ml 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT; Sigma cat. no. M2003‐1G) in phosphate buffered saline (PBS; see recipe in appendix 2A)
  • Lysis buffer (see recipe)
  • Microplate spectrophotometer (e.g., Wallac 1420 Victor2 multilabel counter; PerkinElmer)

Basic Protocol 3: Cell Viability Assay by Colloidal Dye (Trypan Blue) Exclusion

  Materials
  • Cells in 24‐well culture plate (500 µl bathing medium/well)
  • HEPES‐buffered salt solution (HBS, see recipe)
  • 0.4% trypan blue solution (Sigma cat. no. T8154)

Basic Protocol 4: Cell Viability Assay Using Propidium Iodide

  Materials
  • 1 mg/ml propidium iodide (Molecular Probes cat. no. P3566)
  • Cells in 24‐well culture plates (500 µl bathing medium/well)
  • Fluorescence multiwell plate reader (e.g., Wallac 1420 Victor2 multilabel counter; PerkinElmer) with a 530 ± 25 nm excitation filter and a 645 ± 40 nm emission filter, optional
  • Fluorescence microscope, optional

Support Protocol 1: Preservation of Stained Cells

  Materials
  • Stained cells (see protocol 4 or protocol 54)
  • Phosphate buffered saline (PBS; appendix 2A)
  • Phosphate‐buffered 4% paraformaldehyde (FD NeuroTechnologies), ice cold
  • Parafilm and/or aluminum foil

Basic Protocol 5: Transfected Cell Viability Assay by Firefly Luciferase Activity

  Materials
  • Minimal essential media (MEM without phenol red; Invitrogen)
  • Steadylite luciferase assay kit (PerkinElmer cat. no. 6016981) containing:
    • Steadylite HTS substrate solution (lyophilized)
    • Steadylite HTS substrate buffer solution
  • Firefly luciferase (e.g., pGL2, Promega cat. no. E1611)–transfected cells in 24‐well culture plate (see appendix 1F for cationic‐lipid‐based transfection protocols), 24 to 48 hr post‐transfection
  • Repeater pipet
  • 96‐well white opaque plate (Corning Life Sciences)
  • Microplate luminometer (e.g., Wallac 1420 Victor2 multilabel counter; PerkinElmer)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Boeckman, F.A. and Aizenman, E. 1996. Pharmacological properties of acquired excitotoxicity in Chinese hamster ovary cells transfected with N‐methyl‐D‐aspartate receptor subunits. J. Pharmacol. Exp. Ther. 279:515‐523.
   Bozyczko‐Coyne, D., McKenna, B.W., Connors, T.J., and Neff, N.T. 1993. A rapid fluorometric assay to measure neuronal survival in vitro. J. Neurosci. Meth. 50:205‐216.
   Bredesen, D.E., Rao, R.V., and Mehlen, P. 2006. Cell death in the nervous system. Nature 443:796‐802.
   Burdon, R.H., Gill, V., and Rice‐Evans, C. 1993. Reduction of a tetrazolium salt and superoxide generation in human tumor cells (HeLa). Free Radic. Res. Commun. 18:369‐380.
   Hansen, M.B., Nielsen, S.E., and Berg, K. 1989. Re‐examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J. Immunol. Meth. 119:203‐210.
   Hartnett, K.A., Stout, A.K., Rajdev, S., Rosenberg, P.A., Reynolds, I.J., and Aizenman, E. 1997. NMDA receptor‐mediated neurotoxicity: A paradoxical requirement for extracellular Mg2+ in Na+/Ca2+‐free solutions in rat cortical neurons in vitro. J. Neurochem. 68:1836‐1845.
   Hertel, C., Hauser, N., Schubenel, R., Seilheimer, B., and Kemp, J.A. 1996. Beta‐amyloid‐induced cell toxicity: Enhancement of 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide‐dependent cell death. J. Neurochem. 67:272‐276.
   Kerr, J.F.R., Wyllie, A.H., and Currie, A.R. 1972. Apoptosis: A basic biological phenomenon with wide‐ranging implications in tissue kinetics. Br. J. Cancer 26:239‐257.
   Kingsbury, A.E., Gallo, V., Woodhams, P.L., and Balazs, R. 1985. Survival, morphology and adhesion properties of cerebellar interneurons cultured in chemically defined and serum‐supplemented medium. Brain Res. 349:17‐25.
   Koh, J.Y. and Choi, D.W. 1987. Quantitative determination of glutamate‐mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay. J. Neurosci. Meth. 20:83‐90.
   Koh, J.Y. and Choi, D.W. 1988. Vulnerability of cultured cortical neurons to damage by excitotoxins: Differential susceptibility of neurons containing NADPH‐diaphorase. J. Neurosci. 8:2153‐2163.
   Mattson, M.P., Lovell, M.A., Ehmann, W.D., and Markesbery, W.R. 1993. Comparison of the effects of elevated intracellular aluminum and calcium levels on neuronal survival and tau immunoreactivity. Brain Res. 602:21‐31.
   McLaughlin, B., Pal, S., Tran, M.P., Parsons, A.A., Barone, F.C., Erhardt, J.A., and Aizenman, E. 2001. p38 activation is required upstream of potassium current enhancement and caspase cleavage in thiol oxidant‐induced neuronal apoptosis. J. Neurosci. 21:3303‐3311.
   Mills, J.C., Nelson, D., Erecinska, M., and Pittman, R.N. 1995. Metabolic and energetic changes during apoptosis in neural cells. J. Neurochem. 65:1721‐1730.
   Moore, P.L., MacCoubrey, I.C., and Haugland, R.P. 1990. A rapid, pH insensitive, two color fluorescence viability (cytotoxicity) assay. J. Cell. Biol. 111:58a
   Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferase and cytotoxicity assays. J. Immunol. Meth. 65:55‐63.
   Ormerod, M.G., Collins, M.K., Rodriguez‐Tarduchy, G., and Robertson, D. 1992. Apoptosis in interleukin‐3‐dependent haemopoietic cells. Quantification by two flow cytometric methods. J. Immunol. Methods 153:57‐65.
   Pal, S., Hartnett, K.A., Nerbonne, J.M., Levitan, E.S., and Aizenman E. 2003. Mediation of neuronal apoptosis by Kv2 1‐encoded potassium channels. J. Neurosci. 23:4798‐4802.
   Pender, M.P., Nguyen, K.B., McCombe, P.A., and Kerr, J.F. 1991. Apoptosis in the nervous system in experimental allergic encephalomyelitis. J. Neurol. Sci. 104:81‐87.
   Perry, S.W., Epstein, L.G., and Gelbard, H.A. 1997. Simultaneous in situ detection of apoptosis and necrosis in monolayer cultures by TUNEL and trypan blue staining. Biotechniques 22:1102‐1106.
   Pettmann, B. and Henderson, C.E. 1998. Neuronal cell death. Neuron 20:633‐647.
   Santos, S. and Aizenman, E. 2002. Functional expression of muscle‐type nicotinic acetylcholine receptors in rat forebrain neurons in vitro. Meth. Find. Exp. Clin. Pharmacol. 24:63‐66.
   Sinor, J.D., Du, S., Venneti, S., Blitzblau, R.C., Leszkiewicz, D.N., Rosenberg, P.A., and Aizenman, E. 2000. NMDA and glutamate evoke excitotoxicity at distinct cellular locations in rat cortical neurons in vitro. J. Neurosci. 20:8831‐8837.
   Slater, T.F., Sawyer, B., and Straeuli, U. 1963. Studies on succinate‐tetrazolium reductase systems. III. Points of coupling of four different tetrazolium salts. Biochim. Biophys. Acta 77:383‐393.
   Trost, L.C. and Lemasters, J.J. 1994. A cytotoxicity assay for tumor necrosis factor employing a multiwell fluorescence scanner. Anal. Biochem. 220:149‐153.
   Verdaguer, E., Pubill, D., Rimbau, V., Jimenez, A., Escubedo, E., Camarasa, J., Pallas, M., and Camins, A. 2002. Evaluation of neuronal cell death by laser scanning cytometry. Brain Res. Protoc. 9:41‐48.
   Vincent, A.M. and Maiese, K. 1999. Direct temporal analysis of apoptosis induction in living adherent neurons. J. Histochem. Cytochem. 47:661‐672.
Key References
   Koh and Choi, 1987. See above.
  Describes the first use of LDH release as a marker for cell death in cultured cortical neurons.
   Slater et al., 1963. See above.
  These two papers include the initial description of the MTT assay as well as its subsequent development and optimization.
   Hansen et al., 1989. See above.
  Describes the use of a multiwell fluorescence scanner to provide a rapid, automated method for assaying cell viability using propidium iodide.
   Trost and Lemasters, 1994. See above.
  These papers utilize luciferase reporter assays to quantify cell death in transfected neurons and Chinese hamster ovary cells expressing mutant receptors or channels.
   Boeckman and Aizenman, 1996. See above.
   Pal et al., 2003. See above.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library