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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  •   FigureFigure 12.18.1 Cell‐free Mn‐fura‐2 standard curve. A 10‐point Mn‐fura‐2 standard curve was generated using a cell‐free system composed of 0.5 µM fura‐2/extraction buffer/Triton X‐100 buffer at Ex360/Em535. Changes in fura‐2 fluorescence induced by MnCl2 quenching were normalized as percent maximal fluorescence (%MAX) to the 100% maximal fluorescence value after background subtraction. Data is represented as %MAX ( x axis) and log10 scale of MnCl2 concentration ( y axis). To enable back calculation of Mn concentration from %MAX, a power curve (solid black trend line) was used for %MAX values < 50% and a logarithmic curve (dashed black trend line) for values > 50%, on either side of the inflection point of the saturation‐binding curve. Mean levels are indicated as ± standard deviation. N = 4 wells/exposure condition. Dashed vertical lines indicate the optimal detection range of CFMEA (10% to 85% maximal fluorescence).
  •   FigureFigure 12.18.2 Inverse relationship between %MAX and total extracted Mn levels (nM). Wild‐type striatal cells exhibit a concentration‐dependent decrease in % maximal RFU that inversely correlates with the increase in total extracted cellular Mn levels (nM). Wild‐type ST HdhQ7/Q7 striatal cell lines were exposed to different MnCl2 concentrations for 4 hr in culture medium. (A) After Mn exposure, changes in %MAX values for each cell extract were normalized to average RFU of the untreated striatal cells after background subtraction. Data is represented as % maximal RFU normalized to untreated cells ( y axis) and concentration of MnCl2 exposed to cells ( x axis). (B) Total extracted cellular Mn levels (nM) from the striatal cells derived in (A) were calculated using the power and logarithmic equations described in Figure and represented as total extracted Mn levels ( y axis) and concentration of MnCl2 exposed to cells ( x axis). Dashed line represents the minimal accurate detection limit of CFMEA (100 µM Mn). N = 3; 6 wells/exposure condition. Mean levels are indicated as 95% confidence interval.
  •   FigureFigure 12.18.3 PicoGreen standard curve. A seven‐point PicoGreen standard was generated using a cell‐free system composed of a 1:400 dilution of PicoGreen reagent in 1× TE buffer and varying salmon testes dsDNA concentrations at Ex485/Em535. The final concentration of salmon testes dsDNA in each well was calculated and plotted as final concentration of DNA ( x axis) and PicoGreen RFU ( y axis). Data points are represented on a linear curve generated by Microsoft Excel. N = 4 wells/dsDNA concentration. Mean levels are indicated as 95% confidence intervals. Dashed vertical arrow line indicates the calculated concentration of dsDNA in representative unexposed wild‐type striatal cells.
  •   FigureFigure 12.18.4 Normalization of total extracted cellular Mn levels by PicoGreen Assay. Wild‐type striatal cells exhibit a concentration‐dependent increase in net Mn uptake following MnCl2 exposure and normalization by PicoGreen assay. The PicoGreen assay described in Figure was performed on the same wild‐type cell extracts described in Figure . The final DNA concentrations in wild‐type striatal cell extracts were calculated using the PicoGreen standard curve equation generated in Fig. . Total extracted cellular Mn levels in each cell extract were normalized to its respective DNA concentration. Data is represented as femtomoles Mn per µg DNA ( y axis) and concentration of MnCl2 exposed to cells ( x axis). N = 3; 6 wells/exposure condition. Mean levels are indicated as 95% confidence interval.


Literature Cited

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