Solarbio Cell Iron Content Assay Kit 50T | 48S

Cell Iron Content Assay Kit

Note: It is necessary to predict 2-3 large difference samples before the formal determination. Operation Equipment: Spectrophotometer

Cat No: BC5310

Size:50T/48S

Components:

Extract solution: Liquid 30 mL×1. Store at 2-8℃.

Reagent I: Liquid 15 mL×1. Store at 2-8℃. Reagent II: Liquid 35 mL×1. Store at 2-8℃. Reagent III: Liquid 6 mL×1. Store at 2-8℃.

Standard: Liquid 1 mL×1. Store at 2-8℃. 1 μmol/mL of Fe3+ standard solution. Add 200μL distilled water to 200μL Fe3+ standard solution of 1 μmol/mL before use, and mix well to form the Fe3+ standard solution of 0.5 μmol/mL.

Product Description:

Iron is one of the essential trace elements in the human body, which is the main component of hemoglobin, myoglobin, cytochrome, and other enzyme systems. Iron can assist in the transport of oxygen and promote fat oxidation. Iron deficiency can easily cause anemia, and metabolic disorders,  and affect the immune function of the body.

Fe3+ is reduced by hydroxylamine hydrochloride to Fe2+. Fe2+ could react with Phenanthroline Monohydrate to form a kind of orange compound under weak acid conditions, which has an absorption peak at 510 nm. According to the measure, absorbance at 520 nm can reflect cell iron concentration.

Reagents and Equipment Required but Not Provided:

Spectrophotometer, desk centrifuge, adjustable pipette, 1mL glass cuvette, cell ultrasonic crusher, ice, distilled water.

Procedure:

1. Sample preparation:
2. Collect bacteria or cells into the centrifuge tube, and discard the supernatant after centrifugation. According to the proportion of bacteria or cell number (104): Extract solution volume (mL) of 500-1000-1 to extract. It is suggested that 5 million bacteria or cells amount to 0.5mL of Extract solution. Split the bacteria or cell with ultrasonication (placed on ice, ultrasonic power 200W, working time 3s, interval 7s, repeat 30 times). Centrifuge at 8000 g for 10 minutes at 4℃ to remove insoluble materials, and take the supernatant on ice before
3. Ultrasonication working time could be prolonged properly if samples are fungi, bacteria, or other microorganisms with cell

II. Determination procedure:

1. Preheat the spectrophotometer for 30 minutes, adjust the wavelength to 510 nm, and set zero with distilled
2. Add reagents with the following:

III. Cell Iron Content Calculations

• Bacteria/cells number

Cell iron content (ng/104  cell) =(Cs×ΔAT÷ΔAS)×VE×103×55.845÷500=27.922×ΔAT÷ΔAS

2) Protein concentration

Cell iron content (ng/mg prot)

=(Cs×ΔAT÷ΔAS)×VE×103×55.845÷(Cpr×VE)=27922.5×ΔAT÷ΔAS÷Cpr

Cs: Concentration of Fe3+  standard solution, 0.5μmol/mL;

VE: Extract solution volume, 0.5 mL;

103: Unit conversion factor, 1 μmol=103 nmol;

55.845: Relative molecular mass of Fe, 55.845ng/nmol; 500: Total number of bacteria or cells, 5 million;

Cpr: Supernatant sample protein concentration, mg/mL.

Note:

1. If ΔAT＜01, it is recommended to increase the sample supernatant size before determination. If AT＞1,  it is recommended to dilute the sample supernatant with distilled water before determination. And modify the calculation formula.
2. Sample supernatant protein concentration needs to be measured by yourself if cell iron content is calculated by protein

Experimental example:

1. Take 5 million cells, add 0.5 mL of Extract solution, and split with ultrasonication. Centrifuge and take the supernatant. Then operate according to the determination steps, calculate ΔAT=AT-AB= 0.171- 0.000=0.171, ΔAS=AS-AB=0.573-0.000=0.573. The result is calculated according to cell numbers: Cell iron content (ng/104 cells) =27.922×ΔAT÷ΔAS=8.333ng/104 cell

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