title: Cellular fura-2 manganese extraction assay (CFMEA).
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 (MnCl(2)) 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.
authors.affiliation: Vanderbilt University Medical Center, Nashville, Tennessee, USA.
authors.forename: Gunnar F
authors.initials: GF
authors.lastname: Kwakye
authors.forename: Daphne
authors.initials: D
authors.lastname: Li
authors.forename: Olympia A
authors.initials: OA
authors.lastname: Kabobel
authors.forename: Aaron B
authors.initials: AB
authors.lastname: Bowman
mesh_terms: D000818:Animals; D001692:Biological Transport; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D002138:Calibration; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D005456:Fluorescent Dyes; D016257:Fura-2; D057166:High-Throughput Screening Assays / Q000592:standards; D007700:Kinetics; D017895:Manganese Compounds / Q000378:metabolism*; D051379:Mice; D013050:Spectrometry, Fluorescence / Q000592:standards
chemical_list: D002712:Chlorides; D005456:Fluorescent Dyes; D017895:Manganese Compounds; D004247:DNA; C025340:manganese chloride; D016257:Fura-2