Unit

UNIT 12.18 Cellular fura-2 Manganese Extraction Assay (CFMEA)

  1. Gunnar F. Kwakye1,2,
  2. Daphne Li1,2,
  3. Olympia A. Kabobel1,2,
  4. Aaron B. Bowman1,2

Published Online: 1 MAY 2011

DOI: 10.1002/0471140856.tx1218s48

Current Protocols in Toxicology

Current Protocols in Toxicology

How to Cite

Kwakye, G. F., Li, D., Kabobel, O. A. and Bowman, A. B. 2011. Cellular fura-2 Manganese Extraction Assay (CFMEA). Current Protocols in Toxicology. 48:12.18:12.18.1–12.18.20.

Author Information

  1. 1

    Vanderbilt University Medical Center, Nashville, Tennessee

  2. 2

    Vanderbilt Kennedy Center for Research on Human Development, Nashville, Tennessee

Publication History

  1. Published Online: 1 MAY 2011
  2. Published Print: MAY 2011

Abstract

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