Bathophenanthroline Sulfonate-Based Colorimetric Assay: A Simple and Rapid Method for Quantitation of Non-Heme Iron in Mouse Liver Tissue

Transfer each dried piece of tissue into a 1.5-milliliter microcentrifuge tube. Add 1 milliliter of the acid mixture to the tube, and close it. Prepare an acid blank in the same way, except that the tissue is omitted here.

Incubate the tubes at 65 degrees Celsius for 20 hours to digest the tissue.

After cooling to room temperature, use a micropipette fitted with plastic tips to transfer 500 microliters of the clear acid extract into a new 1.5-milliliter microcentrifuge tube.

Prepare chromogen reactions directly into the flat-bottom, 96-well, clear, untreated, polystyrene microplates. Incubate at room temperature for 15 minutes.

Measure sample absorbance in a plate reader at a wavelength of 535 nanometers against a deionized water reference.