Cellular fura‐2 Manganese Extraction Assay (CFMEA)

Gunnar F. Kwakye1, Daphne Li1, Olympia A. Kabobel1, Aaron B. Bowman1

1 Vanderbilt Kennedy Center for Research on Human Development, Nashville, Tennessee
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 12.18
DOI:  10.1002/0471140856.tx1218s48
Online Posting Date:  May, 2011
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Cellular manganese (Mn) uptake and transport dynamics can be measured using a cellular fura‐2 manganese extraction assay (CFMEA). The assay described here uses immortalized murine striatal cell line and primary cortical astrocytes, but the method is equally adaptable to other cultured mammalian cells. An ultrasensitive fluorescent nucleic acid stain for quantification of double‐stranded DNA (dsDNA) in solution, Quant‐iT PicoGreen, has been utilized for normalization of Mn concentration in the cultured cells, following Mn (II) chloride (MnCl2) exposure. Depending on the cell type and density, other methods, e.g., protein determination assays or cell counts, may also be used for normalization. Methods are described for rapidly stopping Mn uptake and transport processes at specified times, extraction, and quantification of cellular Mn content, and normalization of Mn levels to dsDNA concentration. Curr. Protoc. Toxicol. 48:12.18.1‐12.18.20. © 2011 by John Wiley & Sons, Inc.

Keywords: manganese; high‐throughput assay; metal transport; fura‐2

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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of Cellular Manganese Levels
  • Basic Protocol 2: Normalization of Cellular Mn Levels by Quant‐iT PicoGreen Reagent
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Measurement of Cellular Manganese Levels

  • Immortalized murine striatal cell line (E4 striatal primordial cells lines of wild‐type Huntingtin and mutant knock‐in embryos; Trettel et al., ) or primary cortical astrocytes (from rat pups; Aschner et al., ) growing in a 10‐cm2 tissue culture dish in 10 ml DMEM‐10 dish (see recipe)
  • CMF‐DPBS: 1× ultrapure phosphate‐buffered saline, pH 7.4, without calcium and magnesium (e.g., Mediatech cat. no. 21‐040‐CV; also see appendix 2A)
  • DMEM‐10 (see recipe)
  • 0.05% (w/v) trypsin (e.g., Invitrogen cat. no. 253000‐54): store up to 6 months at −20°C
  • 1000× MnCl 2 working dilutions (see recipe)
  • HEPES salt exposure buffer, pH 7.2 ( see recipe)
  • Krebs Ringer buffer (see recipe)
  • 1 mM fura‐2 stock solution (see recipe)
  • Extraction buffer: PBS/0.1% (v/v) Triton X‐100
  • TE buffer (see recipe)
  • Pasteur glass pipet, sterile
  • Laminar flow hood
  • Tissue culture incubator: 5% CO 2, 33°C for immortalized striatal cells lines or 5% CO 2, 37°C for primary cortical astrocytes
  • Hemacytometer or automated cell counter (e.g., Cellometer Auto T4, Nexcelom Bioscience)
  • 96‐well tissue culture plate
  • Multichannel pipettor (Thermo Scientific), recommended
  • Paper towels
  • 50‐ml conical, polypropylene centrifuge tubes, sterile, cell culture–tested
  • Beckman coulter DTX 880 multimode plate reader with multimode analysis software version, or equivalent
  • Beckman coulter DTX 880 multimode excitation filters: 360‐nm excitation (filter bandwidth ±35 nm) and 485‐nm excitation (filter bandwidth ±20 nm), or equivalent
  • Beckman coulter DTX 880 multimode emission filter: 535‐nm emission (filter bandwidth ±25 nm), or equivalent
  • Parafilm
  • Microsoft Excel

Basic Protocol 2: Normalization of Cellular Mn Levels by Quant‐iT PicoGreen Reagent

  • Salmon testes dsDNA standards (see recipe)
  • Quant‐iT PicoGreen dsDNA Reagent, 1 ml (Molecular Probes cat. no. P7589)
  • 1× TE buffer (see recipe)
  • Extraction buffer: CMF‐DPBS ( appendix 2A)/0.1% (v/v) Triton‐X‐100
  • Cell extracts obtained following CFMEA analysis in 96‐well tissue culture plates ( protocol 1)
  • 96‐well tissue culture plates
  • Microcentrifuge tubes
  • Beckman coulter DTX 880 multimode plate reader (multimode analysis software, version or similar device
  • Beckman coulter DTX 880 multimode excitation and emission filters: excitation 485 nm (filter bandwidth ± 20 nm) and emission 535 nm (filter bandwidth ±25 nm)
  • Microsoft Excel
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Literature Cited

Literature Cited
   Ahn, S.J., Costa, J., and Emanuel, J.R. 1996. PicoGreen quantitation of DNA: Effective evaluation of samples pre‐ or post‐PCR. Nucleic Acids Res. 24:2623‐2625.
   Aschner, M., Gannon, M., and Kimelberg, H.K. 1992. Manganese uptake and efflux in cultured rat astrocytes. J. Neurochem. 58:730‐735.
   Aschner, M., Guilarte, T.R., Schneider, J.S., and Zheng, W. 2007. Manganese: Recent advances in understanding its transport and neurotoxicity. Toxicol. Appl. Pharmacol. 221:131‐147.
   Baruthio, F., Guillard, O., Arnaud, J., Pierre, F., and Zawislak, R. 1988. Determination of manganese in biological materials by electrothermal atomic absorption spectrometry: A review. Clin. Chem. 34:227‐234.
   Butterworth, J. 1986. Changes in nine enzyme markers for neurons, glia, and endothelial cells in agonal state and Huntington's disease caudate nucleus. J. Neurochem. 47:583‐587.
   Cobbold, P.H. and Rink, T.J. 1987. Fluorescence and bioluminescence measurement of cytoplasmic free calcium. Biochem. J. 248:313‐328.
   Enger, Ø. 1996. Use of the fluorescent dye PicoGreen for quantification of PCR products after agarose gel electrophoresis. Biotechniques 21:372‐374.
   Erikson, K.M. and Aschner, M. 2003. Manganese neurotoxicity and glutamate‐GABA interaction. Neurochem. Int. 43:475‐480.
   Erikson, K.M., Syversen, T., Aschner, J., and Aschner, M. 2005. Interactions between excessive manganese‐exposure and dietary iron‐deficiency in neurodegeneration. Environ. Toxicol. Pharmacol. 19:415‐421.
   Forbes, J.R. and Gros, P. 2003. Iron, manganese, and cobalt transport by Nramp1 (Slc11a1) and Nramp2 (Slc11a2) expressed at the plasma membrane. Blood 102:1884‐1892.
   Grynkiewicz, G., Poenie, M., and Tsien, R.Y. 1985. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260:3440‐3450.
   Hurley, L.S. and Keen, C.L. 1987. Manganese In Trace Elements in Human Health and Animal Nutrition (E. Underwood and W. Mertz, eds.) pp. 185‐225. Academic Press, New York.
   Kwakye, G.F., Li, D., and Bowman, A.B. 2011. Novel high‐throughput assay to assess cellular manganese levels in a striatal cell line model of Huntington's disease confirms a deficit in manganese accumulation. Neurotoxicology Jan. 14 [Epub ahead of print].
   Lim, D., Feedrizzi, L., Tartari, M., Zuccato, C., Cattaneo, E., Brini, M., and Carafoli, E. 2007. Calcium homeostasis and mitochondrial dysfunction in striatal neurons of Huntington disease. J. Biol. Chem. 283:5780‐5789.
   Merritt, J.E., Jacob, R., and Hallam, T.J. 1989. Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophils. J. Biol. Chem. 264:1522‐1527.
   Molecular Probes. 1996. PicoGreen dsDNA Quantitation Reagent and Kit Instructions. Eugene, Oregon.
   Oliveira, J.M.A., Chen, S., Almeida, S., Riley, R., Goncalves, J., Oliveira, C.R., Hayden, M.R., Nicholls, D.G., Ellerby, L.M., and Christina Rego, A. 2006. Mitochondrial‐dependent Ca2+ handling in Huntington's disease striatal cells: Effect of histone deacetylase inhibitors. J. Neurosci. 26:11174‐11186.
   Picard, V., Govoni, G., Jabado, N., and Gros, P. 2000. Nramp 2 (DCT1/DMT1) expressed at the plasma membrane transports iron and other divalent cations into a calcein‐accessible cytoplasmic pool. J. Biol. Chem. 275:35738‐35745.
   Seville, M., West, A.B., and McHenry, C.S. 1996. Fluorometric assay for DNA polymerases and reverse transcriptase. Biotechniques 21:664‐672.
   Snitsarev, V.A., McNulty, T.J., and Taylor, C.W. 1996. Endogenous heavy metal ions perturb fura‐2 measurements of basal and hormone‐evoked Ca2+ signals. Biophys. J. 71:1048‐1056.
   Tang, T.S., Tu, H., Maximov, A., Wang, Z., Wellington, C.L., Hayden, M.R., and Bezprozvanny, I. 2003. Huntingtin and huntingtin‐associated protein 1 influence neuronal calcium signaling mediated by inositol‐(1,4,5) triphosphate receptor type 1. Neuron 39:227‐239.
   Trettel, F., Rigamonti, D., Hilditch‐Maguire, P., Wheeler, V.C., Sharp, A.H., Persichetti, F., Cattaneo, E., and MacDonald, M.E. 2000. Dominant phenotypes produced by the HD mutation in STHdh(Q111) striatal cells. Hum. Mol. Genet. 9:2799‐2809.
   Tsien, R.Y. 1989. Fluorescent probes of cell signaling. Ann. Rev. Neurosci. 12:227‐253.
   Tsien, R. and Pozzan, T. 1989. Measurement of cytosolic free Ca2+ with quin‐2. Methods Enzymol. 172:230‐262.
   Tsien, R.W., Ellinor, P.T., and Horne, W.A. 1991. Molecular diversity of voltage‐dependent Ca2+ channels. Trends Pharmacol. Sci. 12:349‐354.
   Williams, B.B., Kwakye, G.F., Wegrzynowicz, M., Li, D., Aschner, M., Erikson, K.M., and Bowman, A.B. 2010a. Altered manganese homeostasis and manganese toxicity in a Huntington's disease striatal cell model are not explained by defects in the iron transport system. Toxicol. Sci. 117:169‐179.
   Williams, B.B., Li, D., Wegrzynowicz, M., Vadodaria, B.K., Anderson, J.G., Kwakye, G.F., Aschner, M., Erikson, K.M., and Bowman, A.B. 2010b. Disease‐toxicant screen reveals a neuroprotective interaction between Huntington's disease and manganese exposure. J. Neurochem. 112:227‐237.
   Xu, B., Xu, Z.F., and Deng, Y. 2009. Effect of manganese exposure on intracellular Ca2+ homeostasis and expression of NMDA receptor subunits in primary cultured neurons. Neurotoxicology 30:941‐949.
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