铁是人体必需的元素之一，对人体的新陈代谢和身体健康具有重要作用，血液中铁的含量不足，就会造成缺铁性贫血。铁存在于人体所有的细胞内，各组织、器官包括各内分泌腺都含有铁。肝脏、脾脏和肺组织内含量较为丰富。成人体内含铁3～5克，大部分都以蛋白质复合物形式存在，极少部分以离子的形态存在。本试剂盒可用于动植物组织、细胞和血清（浆）或其他液体中的总铁离子定量。在酸性条件下，铁从蛋白质复合物中解离出来，然后在还原剂的作用下三价的铁离子(Fe³⁺) 被还原为 Fe²⁺。Fe²⁺在酸性条件下与三吡啶基三嗪（TPTZ）形成蓝色配合物，在593nm处有吸收峰，通过测定该波长吸光度即可计算总铁离子的含量。
T1515789
| 组分 | 物理外观 | 48T | 96T | 储存 | 单次测试使用量 | | T1515789A | 缓冲液 | 液体 | 15 mL | 30 mL | 2-8℃. 避光 | 按需 | | T1515789B | 显色液 | 液体 | 7 mL | 14 mL | 2-8℃. 避光 | 100 μL | | T1515789C | 铁标准品（40mM） | 液体 | 0.5 mL | 1 mL | 2-8℃. 避光 | 10 μL | | T1515789D | 提取液 | 液体 | 15 mL | 30 mL | 2-8℃. 避光 | 按需 |
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使用方法
1. 样品处理
(1) 组织样本：按照每0.01 g组织加入0.1 mL提取液的比例，用匀浆器或研钵冰浴匀浆。12000× g，4℃离心 10 min，收集上清液，置冰上待测。
(2) 细胞样本：收集细胞到离心管内，用冷 PBS 清洗细胞，离心后弃上清。按照每100万细胞加入0.1 mL提取液的比例，置冰上裂解 15 min，每 3 min 上下颠倒混匀一次，裂解完成后，12000× g，4℃离心 10 min，置冰上待测。
(3) 血清（浆）等液体样本：取55 μL 样本，加入165 μL缓冲液混合均匀（即稀释 4 倍），置冰上待测。如果样本浑浊，5000× g，4℃离心 5 min，取上清液，置冰上待测。
注：本试剂盒提取液不能使用 BCA 蛋白定量，如需测定蛋白浓度，推荐使用阿拉丁即用型Bradford蛋白定量试剂盒 (去垢剂兼容型) （B1509656）。
2. 样品稀释
(1) 为确保样品数值在标准曲线范围内，建议进行预实验将样品设置多个稀释倍数，以确定样品中总铁离子的大致浓度，如果数值不在标准曲线范围内，请调整样品的稀释倍数。
(2) 血清（浆）等液体样本在样本处理过程中已稀释 4 倍，通常无需再进一步稀释。细胞样本的总铁离子含量较低，也不建议稀释样品。
(3) 样本如需要稀释，血清（浆）等液体样本的稀释建议使用缓冲液，而细胞和组织样品的稀释建议使用提取液。
3. 绘制标准曲线
将40 mmol/L（40000 μmol/L）铁标准品用缓冲液进行稀释得到100、50、25、12.5、6.25、3.125、1.5625 μmol/L的标准品，现配现用。
标准品稀释可参考下表：
| 序号 | 稀释前浓度（μmol/L） | 标准品体积（μL） | 缓冲液体积（μL） | 稀释后浓度（μmol/L） | | 1 | 40000 | 10 | 990 | 400 | | 2 | 400 | 150 | 450 | 100 | | 3 | 100 | 300 | 300 | 50 | | 4 | 50 | 300 | 300 | 25 | | 5 | 25 | 300 | 300 | 12.5 | | 6 | 12.5 | 300 | 300 | 6.25 | | 7 | 6.25 | 300 | 300 | 3.125 | | 8 | 3.125 | 300 | 300 | 1.5625 |
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备注：下述实验中每个标准管需200 μL标准品（注意不要在此步骤直接检测吸光度）。
4. 样品测定
(1) 酶标仪预热 30 min 以上，调节波长到 593 nm。
(2) 按下表设置反应体系加样。设置不加样品的空白对照管。
| 试剂名称 | 空白对照管 | 标准品管 | 测定样品管 | | 样品（μL） | 0 | 0 | 200 | | 标准品（μL） | 0 | 200 | 0 | | 缓冲液（μL） | 200 | 0 | 0 | | 显色液（μL） | 100 | 100 | 100 |
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(3) 混匀，37°C 孵育30 min，若浑浊则需12000× g，离心 10 min后取200 μL上清液于酶标板各对应孔中，在酶标仪593 nm处测定各孔OD值。空白孔的OD值记为A空白，标准品孔的OD值记为A标准，测定样品孔的OD值记为A测定。计算：ΔA测定= A测定 - A空白 ，Δ A标准 = A标准 - A空白 。
5. 总铁离子含量计算
5.1 标准曲线的绘制
以标准品浓度为x轴，Δ A标准 为 y 轴，绘制标准曲线，得到标准方程，将 ΔA测定 带入方程得到样品浓度Cₛₐₘₚₗₑ (μmol/L)。
5.2 总铁离子浓度计算
5.2.1 血清（浆）等液体样本：总铁离子含量 (μmol/L) = Cₛₐₘₚₗₑ × 4* × f；
5.2.2 组织样本：
(1) 以组织湿重进行计算： 总铁离子含量 (μmol/kg wet weight) = Cₛₐₘₚₗₑ × f ÷ (m/V)；
(2) 按蛋白浓度计算：总铁离子含量 (nmol/mg 蛋白) = Cₛₐₘₚₗₑ × f ÷ Cₚᵣ
5.2.3 细胞样本：
(1) 以细胞数量进行计算： 总铁离子含量 (nmol/10⁶ cells ) = Cₛₐₘₚₗₑ × f ÷ (N/V)
(2) 按蛋白浓度计算：总铁离子含量 (nmol/mg 蛋白)= Cₛₐₘₚₗₑ × f ÷ Cₚᵣ
注：
f：样本加入检测体系前的稀释倍数；
4*：血清（浆）等液体样本与缓冲液混合后被稀释了四倍；
m：组织湿重质量（g）；
V：样本提取液加入量(mL)；
Cₚᵣ：样本蛋白质浓度，mg/mL；
N: 用于裂解的细胞样本数量/10⁶ 。
注意事项
1. 操作过程中，样品与试剂等避免与铁器接触，以防污染。
2. 建议使用新鲜样本。如果不立即进行实验，样本可在-80℃保存 1 个月。
3. 所测样本的值高于标准曲线的上限，应稀释样品后重新测定。
4. 正式检测前，建议选择 2-3 个预期差异较大的样本进行预实验。
5. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
结果展示
典型标准曲线：
Iron is one of the essential elements for the human body and plays a vital role in metabolism and overall health. Insufficient iron in the blood can lead to iron deficiency anemia. Iron is present in all cells of the human body, and various tissues and organs, including endocrine glands, contain iron. The liver, spleen, and lung tissues are relatively rich in iron. The adult human body contains 3–5 grams of iron, most of which exists in the form of protein complexes, with only a small fraction in ionic form. This kit can be used for the quantification of total iron ions in animal and plant tissues, cells, serum (plasma), or other liquids. Under acidic conditions, iron is released from protein complexes, and then trivalent iron ions (Fe³⁺) are reduced to Fe²⁺ by a reducing agent. Fe²⁺ forms a blue complex with tripyridyltriazine (TPTZ) under acidic conditions, which exhibits an absorption peak at 593 nm. The total iron ion content can be calculated by measuring the absorbance at this wavelength.
T1515789
| Component | Appearance | 48T | 96T | Storage | Quantity Per Test | | T1515789A | Buffer Solution | Liquid | 15 mL | 30 mL | 2-8℃. Store in the dark | As needed | | T1515789B | Chromogenic Solution | Liquid | 7 mL | 14 mL | 2-8℃. Store in the dark | 100 μL | | T1515789C | Iron Standard（40mM) | Liquid | 0.5 mL | 1 mL | 2-8℃. Store in the dark | 10 μL | | T1515789D | Extraction Buffer | Liquid | 15 mL | 30 mL | 2-8℃. Store in the dark | As needed
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Protocol
1. Sample Preparation
(1) Tissue samples: Add 0.1 mL of Extraction Solution per 0.01 g of tissue, and homogenize using a homogenizer or mortar and pestle on ice. Centrifuge at 12,000× g and 4 °C for 10 minutes, collect the supernatant, and keep it on ice until analysis.
(2) Cell samples: Collect cells into a centrifuge tube, wash with cold PBS, and discard the supernatant after centrifugation. Add 0.1 mL of Extraction Solution per 1 × 10⁶ cells, and lyse on ice for 15 minutes, with mixing by inverting every 3 minutes. After lysis, centrifuge at 12,000× g and 4 °C for 10 minutes, and keep the supernatant on ice for testing.
(3) Serum, plasma, and other liquid samples: Take 55 μL of the sample, add 165 μL of Buffer Solution, and mix thoroughly (i.e., a 4‑fold dilution). Keep on ice until analysis. If the sample is turbid, centrifuge at 5,000× g and 4 °C for 5 minutes, collect the supernatant, and keep on ice for testing.
Note: The Extraction Solution provided in this kit is not compatible with BCA protein assays. If protein concentration determination is required, it is recommended to use Aladdin’s Ready‑to‑Use Bradford Protein Assay Kit (Detergent Compatible) (B1509656).
2. Sample Dilution
(1) To ensure that the sample values fall within the standard curve range, it is recommended to perform a preliminary experiment with multiple dilution factors to determine the approximate concentration of total iron ions in the sample. If the values are not within the standard curve range, adjust the dilution factor accordingly.
(2) Serum, plasma, and other liquid samples have already been diluted 4‑fold during sample processing and usually do not require further dilution. Cell samples generally have low total iron ion content; dilution is also not recommended.
(3) If dilution is necessary, use Buffer Solution for serum/plasma and other liquid samples, and use Extraction Buffer for cell and tissue samples.
3. Preparation of Standard Curve
Dilute the 40 mmol/L (40,000 μmol/L) iron standard with Buffer Solution to obtain standards at concentrations of 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 μmol/L. Prepare fresh immediately before use.
Refer to the table below for standard dilution:
| No. | Pre‑dilution Conc. (μmol/L) | Std. Volume (μL) | Buffer Solution Volume (μL) | Final Conc. (μmol/L) | | 1 | 40000 | 10 | 990 | 400 | | 2 | 400 | 150 | 450 | 100 | | 3 | 100 | 300 | 300 | 50 | | 4 | 50 | 300 | 300 | 25 | | 5 | 25 | 300 | 300 | 12.5 | | 6 | 12.5 | 300 | 300 | 6.25 | | 7 | 6.25 | 300 | 300 | 3.125 | | 8 | 3.125 | 300 | 300 | 1.5625 |
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Note: Each standard tube requires 200 μL of diluted standard for the subsequent assay (do not measure absorbance at this step).
4. Sample Measurement
(1) Pre‑warm the microplate reader for at least 30 min. Set the wavelength to 593 nm.
(2) Prepare the reaction mixtures as shown below. Include a blank control (no sample).
| Reagent | Blank | Standard | Sample | | Sample (μL) | 0 | 0 | 200 | | Standard (μL) | 0 | 200 | 0 | | Buffer Solution (μL) | 200 | 0 | 0 | | Chromogenic Solution (μL) | 100 | 100 | 100 |
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(3) Mix thoroughly and incubate at 37°C for 30 minutes. Transfer 200 μL of the supernatant to the corresponding wells of a microplate. Measure the absorbance of each well at 593 nm. Record the OD value of the blank absorbance as Abₗₐₙₖ, the OD value of standard as Aₛₜₐₙdₐᵣd, and the OD value of sample as Aₛₐₘₚₗₑ. Calculate: ΔAₛₐₘₚₗₑ = Aₛₐₘₚₗₑ – Abₗₐₙₖ , ΔAₛₜₐₙdₐᵣd = Aₛₜₐₙdₐᵣd – Abₗₐₙₖ.
5. Calculation of Total Iron Ion Content
5.1 Preparation of the standard curve: Plot the standard concentration (x‑axis) against ΔAₛₜₐₙdₐᵣd (y‑axis) to obtain the linear equation. Substitute ΔAₛₐₘₚₗₑ into the equation to calculate the sample concentration Cₛₐₘₚₗₑ (μmol/L).
5.2 Calculation of total iron ion concentration
5.2.1 For serum/plasma or other liquid samples: Total iron ion content (μmol/L) = Cₛₐₘₚₗₑ × 4* × f
5.2.2 For tissue samples:
(1) Based on tissue wet weight: Total iron ion content (μmol/kg wet weight) = Cₛₐₘₚₗₑ × f ÷ (m / V)
(2) Based on protein concentration: Total iron ion content (nmol/mg protein) = Cₛₐₘₚₗₑ × f ÷ Cₚᵣ
5.2.3 For cell samples:
(1) Based on cell number: Total iron ion content (nmol/10⁶ cells) = Cₛₐₘₚₗₑ × f ÷ (N / V)
(2) Based on protein concentration: Total iron ion content (nmol/mg protein) = Cₛₐₘₚₗₑ × f ÷ Cₚᵣ
Note:
f: dilution factor before measurement;
4*: Serum, plasma, and other liquid samples are diluted four‑fold after mixing with the Buffer Solution.
m: tissue wet weight (g);
V: volume of Extraction Buffer added (mL);
Cₚᵣ: protein concentration of the sample (mg/mL);
N: number of cells used for lysis (in units of 10⁶).
Precautions:
1. During the procedure, avoid contact of samples and reagents with iron‑containing materials to prevent contamination.
2. Fresh samples are recommended. If the assay is not performed immediately, samples can be stored at –80 °C for up to 1 month.
3. If the measured value of a sample exceeds the upper limit of the standard curve, the sample should be diluted and re‑assayed.
4. Before the formal assay, it is recommended to perform a preliminary experiment using 2‑3 samples that are expected to show significant differences.
5. For your safety and health, please wear a lab coat and disposable gloves during operation.
Results Presentation
Typical standard curve:
ZJ26F0838247
危险品化学品经营许可证（带存储）