TITLE: Colorimetric Iron Quantification Assay
AUTHORS: Natalie Rubio

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Measures iron from 0.4 - 20 nmol/83366) to quantify the levels of iron in cell culture and meat samples. Iron dissociates from its carrier protein in the presence of acidic buffer. When reduced to Fe</span><span style = "vertical-align:super;">2+</span><span>, ions will react with Ferene S to produce a complex with absorbance at 593 nm. Cu</span><span style = "vertical-align:super;">2+</span><span> interference is blocked with a chelate chemical included in the buffer. This protocol is designed to measure iron from 0.4 - 20 nmol/50 μL sample or 8 μM - 400 μM.</span></div></div>

[STEPS]
?. [Prepare the reagents from the Iron Assay Kit.]
1. Equilibrate the iron standard, iron assay buffer and iron reducer to room temperature. 2. Thaw the iron probe and then keep on ice during the duration of the assay.
?. [Prepare the standard curve.]
1. Dilute 10 μL of iron standard in 990 μL of distilled water to prepare a 1 mM standard. Prepare a standard curve dilution in a 96-well plate.  ABCDE1Standard No.1 mM Iron Standard Volume (μL)Assay Buffer Volume (μL)Final Volume (μL)Iron Concentration (nmol/well)210300100032629410024312288100454182821006652427610087630270100102. Set up the dilution for a duplicate reading (2 x 100 μL).
ABCDE1Standard No.1 mM Iron Standard Volume (μL)Assay Buffer Volume (μL)Final Volume (μL)Iron Concentration (nmol/well)21030010003262941002431228810045418282100665242761008763027010010
?. [Prepare the samples.]
1. Use fresh samples or thaw snap frozen samples on ice. 2. Weigh 10 mg of tissue sample for each condition.3. Wash the tissue in cold PBS.4. Homogenize each tissue in 300 μL iron assay buffer with a Dounce homogenizer on ice with 15 passes.5. Transfer the homogenates from the homogenizer to microcentrifuge tubes.6. Centrifuge the homogenates at 16,000 xg for 10 minutes to remove insoluble particles. 7. Transfer the supernatants to clean microcentrifuge tubes and keep on ice.
?. [Perform the assay.]
1. Add 100 μL of standard dilutions in duplicate to a 96-well plate.2. Add 50 μL of each sample in quadruplicate (duplicate for Fe2+, duplicate for total Fe) to the 96-well plate and add 50 μL iron assay buffer to each sample to bring the total volume to 100 μL per well.3. Add 5 μL of iron reducer to each standard well.4. For Fe2+ measurement, add 5 μL of assay buffer to each sample.5. For total Fe measurement, add 5 μL of iron reducer to each sample.6. Mix and incubate standards and samples at 25°C for 30 minutes. 7. Add 100 μL iron probe to each well containing the iron standard and test samples.8. Mix and incubate at 25°C for 60 minutes, in the dark. 9. Measure output on a colorimetric microplate reader at OD 593.
?. [Calculate iron concentrations.]
1. Average the duplicate reader for each standard and sample.2. Substrate the mean absorbance value of the blank (standard no. 1) from all standard and sample readings to obtain the corrected absorbance.3. Plot the corrected absorbance values for each standard as a function of the final iron concentration.4. Calculate the line of best fit.5. Determine Fe2+ and total Fe from the standard curve.6. Calculate Fe3+ by subtracting Fe2+ from the total Fe.7. Iron concentration is calculated as: [C] = (Sa/Sv)*D        [C] = iron concentration        Sa = content of iron in well calculated from standard curve (nmol)        Sv = volume of sample added into the reaction wells (μL)        D = sample dilution factor (if necessary)        (The molecular weight of iron is 55.845 g/mol.)
y = 0.0826x + 0.0199, R2 = 0.9987