59fecl3

Manufactured by PerkinElmer

Sourced in Italy, United States

59FeCl3 is a radioactive iron compound. It is used as a tracer in various scientific applications, including medical research and environmental studies.

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Radiolabeled 59FeCl3 (2 citations)

5 protocols using 59fecl3

Preparation and Characterization of Iron-Humic Complexes

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Water extractable humic substances (WEHS) were isolated as reported by Pinton et al. [64 (link)] and Fe-WEHS complexes were prepared as described by Cesco et al. [31 (link)] by mixing 5 μg organic carbon (Corg) of WEHS fraction for each μmol of FeCl₃. A thorough chemical characterization of the fractions is described elsewhere [23 (link)].
Phytosiderophores (PS) were collected in the root exudate of Fe-deficient barley plants as described by Tomasi et al. [22 (link)]. Iron-PS and Fe-citrate were prepared accordingly to von Wirén et al. [65 (link)] by mixing an aliquot of Fe-free-PS or citrate (10 % excess of the chelating agent) with FeCl₃. For radiochemical experiments, ⁵⁹FeCl₃ was utilized at the specific labeling activity of 144 kBq μmol⁻¹ Fe (Perkin Elmer, Monza, Italy).

Zamboni A., Zanin L., Tomasi N., Avesani L., Pinton R., Varanini Z, & Cesco S. (2016). Early transcriptomic response to Fe supply in Fe-deficient tomato plants is strongly influenced by the nature of the chelating agent. BMC Genomics, 17, 35.

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Optimizing Cellular Iron and Manganese Uptake

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Tet system-suited fetal bovine serum (FBS) was obtained from Clontech (Takara Bio Europe, Saint-Germain-en-Laye, France), Fisher Scientific (Schwerte, Germany) or Atlanta Biological (Atlanta, GA), hygromycin from Carl Roth (Karlsruhe, Germany) or Invitrogen (Carlsbad, CA), and Geneticin (G418) from Biochrom (Berlin, Germany) or Invitrogen (Carlsbad, CA). PGSK diacetate (Molecular Probes) was purchased from Fisher Scientific (Schwerte, Germany). Rotenone (Calbiochem) was from Merck Millipore (Darmstadt, Germany), and 2-(3-carbamimidoylsulfanylmethyl-benzyl)-isothiourea (CISMBI), poly-D-Lysine hydrobromide, doxycycline hyclate, sodium succinate, ferrous sulphate, ferric ammonium citrate (FAC), manganese(II) sulphate, iron(III) chloride, and protease inhibitor cocktail were obtained from Sigma-Aldrich (Taufkirchen, Germany or St. Louis, MO). XEN602 and recombinant human erythropoietin were generously provided by Xenon (Burnaby, BC, Canada) and Amgen (Thousand Oaks, CA), respectively. ⁵⁹FeCl₃ and ⁵⁴MnCl₂ were from Perkin-Elmer (Waltham, MA). ⁵⁷Fe- FAC was from Cambridge Isotope Laboratories, Inc., Andover, MA

Wolff N.A., Garrick M.D., Zhao L., Garrick L.M., Ghio A.J, & Thévenod F. (2018). A role for divalent metal transporter (DMT1) in mitochondrial uptake of iron and manganese. Scientific Reports, 8, 211.

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Cellular Iron Uptake Assay

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Cells were washed three times with uptake buffer (PIB without Ca²⁺ and Mg²⁺), then incubated in uptake buffer for 15 min at 37°C followed by an additional wash to remove endogenous Tf. ⁵⁹FeCl₃ (Perkin Elmer, Waltham, MA) was mixed with citrate (at a ratio of 1: 200) in uptake buffer to make a ⁵⁹Fe³⁺-citrate stock. ⁵⁹Fe³⁺-citrate was added in uptake buffer with (⁵⁹Fe²⁺-citrate) or without (⁵⁹Fe³⁺-citrate) 5 mM ascorbate. For TBI uptake, ⁵⁹FeCl₃ was loaded into apo-transferrin (Sigma-Aldrich, St. Louis, MO) following a published method (McCarthy & Kosman 2012 ); ⁵⁹Fe³⁺-transferrin was added to each cell culture to a final [Tf] = 0.5 μM (1 μM total Fe). Cells were incubated at 37°C for 20 min for NTBI uptake, or 1 hour for TBI uptake, unless noted. Cells were washed four times using ice-cold quenching buffer (uptake buffer containing 1 mM EDTA, pH7.4 for NTBI uptake, pH5.5 for TBI uptake) and harvested in RIPA buffer (50 mM Tris, 150 mM NaCl, 1.0% IGEPAL^® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, pH 8.0.). ⁵⁹Fe accumulated in each sample was measured using an LKB Wallac CompuGamma counter. The amount of ⁵⁹Fe uptake in each sample was quantified and normalized to protein content as determined by BCA assay.

Ji C, & Kosman D.J. (2015). Molecular mechanisms of non-transferrin-bound and transferring-bound iron uptake in primary hippocampal neurons. Journal of neurochemistry, 133(5), 668-683.

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Neuronal Iron Homeostasis Evaluation

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⁵⁹FeCl₃ (1 µM, Perkin Elmer, Waltham, MA) complexed with 4 µM NTA was incubated with neurons in complete Neurobasal medium for 16 h at 37 °C. After ⁵⁹Fe loading, cells were washed thoroughly with DMEM medium containing 20 mM HEPES (pH 7.4) and 1 mM citrate. A set of neurons, counted as “t = 0” or total-accumulated ⁵⁹Fe, were lysed; fresh efflux medium (DMEM medium containing 20 mM HEPES) was added to another set of neurons, which were then incubated at 37 °C. An aliquot of medium was removed from these cultures every 2 h over a 6-h efflux period. ⁵⁹Fe efflux was terminated by washing cells twice with an ice-cold EDTA-containing quench buffer. These t = 6 h cells were lysed. Both medium- and cell-associated ⁵⁹Fe were quantified using an LKB Wallac CompuGamma instrument. The quantity of ⁵⁹Fe was normalized to total protein in each sample as determined by BCA assay.

Ji C., Steimle B.L., Bailey D.K, & Kosman D.J. (2017). The Ferroxidase Hephaestin But Not Amyloid Precursor Protein is Required for Ferroportin-Supported Iron Efflux in Primary Hippocampal Neurons. Cellular and molecular neurobiology, 38(4), 941-954.

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Cellular Uptake of Radioactive Iron-Transferrin

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Radioactive iron-transferrin (⁵⁹Fe₂-Tf) was made from ⁵⁹FeCl₃ (PerkinElmer, Santa Clara, CA, USA; 2 μCi) as described previously.^{28 (link)} For measurements of total cellular ⁵⁹Fe uptake, cells were incubated with 2 μM (final concentration; saturating) ⁵⁹Fe₂-Tf for 3 h, following which samples were washed twice with ice-cold phosphate-buffered saline and collected by centrifugation (200 × g for 5 min at 4°C). The ⁵⁹Fe in heme and non-heme fractions was measured as described in the Online Supplementary Methods.

Garcia-Santos D., Schranzhofer M., Bergeron R., Sheftel A.D, & Ponka P. (2017). Extracellular glycine is necessary for optimal hemoglobinization of erythroid cells. Haematologica, 102(8), 1314-1323.

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