Solarbio Cell Iron Content Assay Kit 50T | 48S

Cell Iron Content Assay Kit

Nota: È necessario prevedere 2-3 campioni con grandi differenze prima della determinazione formale. Attrezzatura operativa: Spettrofotometro

Gatto n: BC5310

Misurare:50T/48S

Componenti:

Estrarre la soluzione: Liquido 30 ml×1. Store at 2-8℃.

Reagente I: Liquido 15 ml×1. Store at 2-8℃. Reagente II: Liquido 35 ml×1. Store at 2-8℃. Reagente III: Liquido 6 ml×1. Store at 2-8℃.

Standard: Liquido 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.

Descrizione del prodotto:

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.

Reagenti e attrezzature necessari ma non forniti:

Spettrofotometro, centrifuga da tavolo, pipetta regolabile, 1Cuvetta di vetro da ml, cell ultrasonic crusher, ghiaccio, acqua distillata.

Procedura:

1. preparazione del campione:
2. Raccogliere i batteri o le cellule nella provetta da centrifuga, and discard the supernatant after centrifugation. According to the proportion of bacteria or cell number (104): Estrarre il volume della soluzione (ml) Di 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 (posto sul ghiaccio, potenza ultrasuoni 200W, Tempo di lavoro 3s, intervallo 7s, ripetere 30 volte). Centrifugare a 8000 g per 10 minuti a 4℃ per rimuovere materiali insolubili, and take the supernatant on ice before
3. Ultrasonication working time could be prolonged properly if samples are fungi, batteri, or other microorganisms with cell

II. Determinazione procedura:

1. Preriscaldare lo spettrofotometro per 30 minuti, regolare la lunghezza d'onda su 510 nm, and set zero with distilled
2. Add reagents with the following:

Reagente (μL)	Provetta (A)	Tubo vuoto (AB)	Tubo standard (COME)
Campione	100	–	–
Acqua distillata	–	100	–
Soluzione standard (0.5 µmol/ml)	–	–	100
Reagente I	200	200	200
Reagente Ⅱ	600	600	600
Reagente Ⅲ	100	100	100
Mescolare accuratamente, and place at 25℃ for 10 minuti. Take 1mL of reaction solution in 1mL glass cuvette. Misurare l'assorbanza a 510 nm, recorded as AT, AB, and AS. ΔAT = AT-AB, ΔAS=AS-AB. Blank tubes and standard tubes only need to be tested once or twice.

III. Cell Iron Content Calcoli

• Bacteria/cells number

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

2) Proteina concentrazione

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: Estrarre il volume della soluzione, 0.5 ml;

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

55.845: Relative molecular mass of Fe, 55.845ng/nmol; 500: Numero totale di batteri o cellule, 5 milioni;

Cpr: Concentrazione di proteina del campione di surnatante, mg/ml.

Nota:

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

Esempio sperimentale:

1. Prendere 5 milioni di cellule, aggiungere 0.5 ml di soluzione di estratto, 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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