BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
铁是人体必需的微量元素之一，也是人体中含量最多的微量元素。铁离子通常以三价铁形式与转铁蛋白结合，通过转铁蛋白通道进入细胞，被金属还原酶STEAP3还原成二价铁后，参与后续多种生理和生化过程。机体内铁元素的缺乏会引发各种缺铁性疾病，如贫血、免疫力低下等，此外还会导致消化不良、记忆力低下等消化系统和神经系统疾病。然而，尽管铁元素对于机体如此重要，却也不是越多越好的。
进入细胞的铁被还原成亚铁离子后，会优先形成各种铁结合复合物，参与到各项生理和生化反应中去。当这些复合物含量接近饱和时，多余的二价铁会在细胞中积累，形成不稳定铁池。铁池中游离的二价铁参与芬顿反应，生成以羟基自由基为代表的活性氧（ROS）物质，积累的ROS将膜脂过氧化，从而造成细胞功能丧失和细胞死亡。这是细胞铁死亡的经典调控途径之一。
动物组织或细胞提取物中非血红素铁含量的多少，反应出细胞或组织贮存铁含量的变化。非血红素铁含量的测定可避免总铁含量测定中样品内血红素铁的影响。
非血红素铁检测试剂盒与总铁测定方法相似，但样品须经酸和热处理，经离心后非血红素铁释放到上清液中，而含血红素铁的蛋白则留在沉淀中。其测定原理是动物组织或细胞经组织消化液在酸性加热条件处理后，经离心可分离出血红素铁和非血红素铁，非血红素铁再被还原剂还原为二价铁后与红菲咯啉生成红色化合物，在500~550nm有强吸收，可通过酶标仪或分光光度计测定。该试剂仅用于科研领域，不适用于临床诊断或其他用途。
产品组成：
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自备材料：
1、蒸馏水或PBS、分光光度计、比色皿、恒温箱或水浴锅；
1、精密天平、离心管、剪刀、震荡器或摇床、离心机、试管、匀浆器。
1、1.5ml 离心管称重。称取 100mg 左右的组织，放入已称重的离心管中，再次称重。两次相减得到组织净重。
2、制备样品消化液：向离心管中加入组织消化液 1ml，65℃消化不少于 50h，中间用震荡器震荡 3~5 次，每次不少于 10min，确保组织完全消化。用组织消化液定容至 1.5ml，10000rpm 离心 5min，将管壁及盖子上的液体甩下，将上清液转移至新离心管，并定容至 1.5ml，混合备用。
3、细胞样品的匀浆和消化：培养细胞经过消化并计数 0.5~1×10⁶/ml，加入等体积的蒸馏水或 PBS 匀浆，匀浆液与组织消化液按 1:5 比例混合，90℃消化 30min，确保细胞完全消化。冷却后 10000rpm 离心 5min，将管壁及盖子上的液体甩下，将上清液转移至新离心管，备用。
4、配制铁显色工作液：将菲啰啉溶液（试剂 C1）、TGA 溶液（试剂 C2）、乙酸钠溶液（试剂 C3）按照 3:3:5 配比得到铁显色工作液，需现配现用。
5、配制系列铁标准液：将铁标准储液 (100μg/ml) 用蒸馏水稀释至 20μg/ml，再按下表进行操作，依次稀释。
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6、加样：取一个无铁污染的干净离心管按照下表设置空白管、标准管、测定管，溶液应按照顺序依次加入，并注意避免产生气泡。如果样品中的铁含量过高，可以减少样品用量或适当稀释后再进行测定，样品的检测最好能设置2平行管，求平均值。
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7、测定：充分混匀，室温显色 10 min。以空白管 1 调零，以分光光度计在 505~550 nm 处测定空白管2、系列标准管、测定管的吸光度，分别记为 A空白 2、A标准、A测定。
计算：
以系列铁标准 (1~5号) (2、5、10、15、20 μg/ml) 为横坐标，以对应的吸光度为纵坐标，绘制标准曲线，求得回归方程，以测定孔的吸光度代入回归方程求得样品消化液中的铁浓度，亦可不做标准梯度，直接进行单标准比色测定，一般选择5或者10 μg/ml 的铁标准。
组织铁含量 (μg/g) = C测定 × V / w = A测定 / A标准 × V / w × C标准
细胞铁含量 (μg / 百万) = C测定 × V × (n / 106) = A测定 / A标准 × V × C标准 × n / 10⁶
式中：
C测定 = 经标曲计算得出的样品消化液中的铁浓度 (μg/mL)；
V = 样品消化液的总体积；
w = 组织重量 (g)；
C标准 = 铁标准浓度 = 5 或 10 (μg/mL)； n = 细胞数量。
注意事项：
1、操作中的手术器械、枪头、容器、比色皿等，一定保证干燥且干净，避免铁污染。
2、组织非血红素铁差异较小，一定每个组织和管子都称重，国产管子的重量相差很多。
3、试剂盒是否过期验证：取10ul铁标准储液(100μg/ml)加入200ul铁显色工作液，看是否显色为深红色，如是，则试剂没过期，可继续使用。
4、试剂有效性判读：用空白管1作对照调零，如空白管2读数＜0.004说明没有污染可以使用，如果空白管2读数＞0.004，说明组织消化液有污染。
5、组织消化液有强酸性和腐蚀性，请小心操作。
6、为了您的安全和健康，请穿实验服并戴一次性手套操作。
7、试剂开封后请尽快使用，以防影响后续实验效果。
有效期：6个月有效，低温运输，按要求保存。
Iron is one of the essential trace elements for the human body and also the most abundant trace element in the body. Iron ions usually bind to transferrin in the form of ferric iron (Fe³⁺), enter cells through transferrin channels, and are reduced to ferrous iron (Fe²⁺) by the metal reductase STEAP3 before participating in various subsequent physiological and biochemical processes. Iron deficiency in the body can cause various iron‑deficiency diseases, such as anemia and low immunity. In addition, it can also lead to digestive system and nervous system disorders such as indigestion and poor memory. However, despite its importance for the body, more iron is not always better.After entering cells, iron is reduced to ferrous ions and preferentially forms various iron‑binding complexes to participate in physiological and biochemical reactions. When these complexes approach saturation, excess ferrous iron accumulates in cells, forming a labile iron pool. Free ferrous iron in the pool participates in the Fenton reaction, generating reactive oxygen species (ROS) represented by hydroxyl radicals. Accumulated ROS causes lipid peroxidation, leading to loss of cellular function and cell death. This is one of the classic regulatory pathways for ferroptosis.The non‑heme iron content in animal tissue or cell extracts reflects changes in stored iron levels in cells or tissues. Determination of non‑heme iron avoids interference from heme iron in samples during total iron measurement.The non‑heme iron assay kit follows a similar principle to total iron determination, but samples require acid and heat treatment. After centrifugation, non‑heme iron is released into the supernatant, while heme iron‑containing proteins remain in the precipitate. The principle is as follows: animal tissues or cells are treated with tissue digestion solution under acidic and heated conditions, and centrifuged to separate heme iron from non‑heme iron. Non‑heme iron is then reduced to ferrous iron by a reducing agent and reacts with bathophenanthroline to form a red compound with strong absorbance at 500~550 nm, which can be measured using a microplate reader or spectrophotometer. This reagent is for research use only and is not intended for clinical diagnosis or other purposes.
Kit Components
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Materials Required but Not Provided
Procedure (for reference only)
1. Weigh a 1.5 mL centrifuge tube. Weigh approximately 100 mg of tissue, place it in the pre‑weighed tube, and weigh again. Subtract the two weights to obtain the net tissue weight.
2. Sample digestion: Add 1 mL of Tissue Digestion Solution to the tube. Digest at 65 °C for no less than 50 h, shaking 3~5 times during incubation, no less than 10 min each time, to ensure complete digestion. Bring the volume to 1.5 mL with Tissue Digestion Solution. Centrifuge at 10,000 rpm for 5 min. Spin down liquid on the tube wall and cap. Transfer the supernatant to a new tube, bring the volume to 1.5 mL, and mix for later use.
3. Cell homogenization and digestion: Harvest cultured cells and adjust the concentration to 0.5~1×10⁶/mL. Homogenize with an equal volume of distilled water or PBS. Mix the homogenate with Tissue Digestion Solution at a ratio of 1:5. Digest at 90 °C for 30 min to ensure complete digestion. Cool and centrifuge at 10,000 rpm for 5 min. Spin down liquid on the tube wall and cap. Transfer the supernatant to a new tube for later use.
4. Preparation of iron chromogenic working solution: Mix Phenanthroline Solution (Reagent C1), TGA Solution (Reagent C2), and Sodium Acetate Solution (Reagent C3) at a ratio of 3:3:5 to prepare the working solution. Prepare fresh before use.
5. Preparation of iron standard series: Dilute Iron Standard Stock Solution (100 μg/mL) with distilled water to 20 μg/mL, then prepare serial dilutions as shown below.
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6. Loading: Use an iron‑free 96‑well microplate. Set blank, standard, and sample wells as shown below. Add solutions in order and avoid bubbles. If iron content is too high, reduce sample volume or dilute. Run samples in duplicate for averaging.
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7. Measurement: Mix thoroughly and develop color at room temperature for 10 min. Zero with Blank 1. Measure absorbance of Blank 2, standards, and samples at 505~550 nm using a spectrophotometer. Record as A<sub>Blank2</sub>, A<sub>Standard</sub>, A<sub>Sample</sub>.
Calculation
Plot a standard curve using iron concentrations (2, 5, 10, 15, 20 μg/mL) as the x‑axis and corresponding absorbance as the y‑axis. Calculate the regression equation and substitute sample absorbance to obtain iron concentration. Alternatively, perform single‑standard colorimetry using 5 or 10 μg/mL standard.
Tissue iron content (μg/g) = CSample × V / w = ASample / AStandard × V / w × CStandard
Cell iron content (μg/10⁶ cells) = CSample × V × (n / 106) = ASample / AStandard × V × CStandard × n / 10⁶
Where:
CSample = iron concentration in sample digest (μg/mL)
V = total volume of sample digest
w = tissue weight (g)
CStandard = iron standard concentration = 5 or 10 (μg/mL)
n = number of cells
Precautions
Shelf Life
6 months when stored as recommended. Transport under cold conditions.
| N1515874 | 组件 | 物理外观 | 50T | 储存 |
| N1515874A | 铁标准储液 (100μg/ml) | 液体 | 2ml | 2-8℃. |
| N1515874B | 组织消化液 | 液体 | 100ml | 2-8℃. 避光 |
| N1515874C | 菲啰啉溶液 | 液体 | 30ml | 2-8℃. 避光 |
| N1515874D | TGA溶液 | 液体 | 30ml | 2-8℃. 避光 |
| N1515874E | 乙酸钠溶液 | 液体 | 50ml | 2-8℃. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0636633 |
分析证书 | N1515874 |
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