产品介绍
L-乳酸在乳酸氧化酶的作用下和氧气发生氧化反应生成丙酮酸盐和H₂O₂。在辣根过氧化物酶存在的情况下，Amplex Red能与过氧化氢反应，产生强烈的红色荧光物质试卤灵，最大激发波长为571 nm，最大发射波长为585 nm，并且在激发波长处有很强的可见光吸收。因此本试剂盒可以用吸光度和荧光两种方法来进行检测, 试卤灵的荧光强度或吸光度与样品中L-乳酸的含量成正比。
产品组分
L1523443 | 组分名称 | 物理外观 | 100 T | 储存 | 单次测试使用量 |
L1523443A | 裂解液 | 液体 | 20 mL | -20℃. | 按需 |
L1523443B | 检测缓冲液 | 液体 | 20 mL | -20℃. | 按需 |
L1523443C | 乳酸氧化酶 | 液体 | 200 μL | -20℃.避光 | 2 μL |
L1523443D | Amplex Red | 液体 | 200 μL | -20℃.避光 | 2 μL |
L1523443E | 辣根过氧化物酶 | 液体 | 200 μL | -20℃.避光 | 2 μL |
L1523443F | L-乳酸标准品 (5 mM) | 液体 | 500 μL | -20℃.避光 | 8 μL |
使用方法
1. 样本处理
(1) 血清 (浆)样品：直接测定。
(2) 细胞样品：每100万细胞加入200 μL裂解液，冰浴5-10分钟或冰浴超声破碎细胞。4ºC，12,000×g离心5分钟，取上清用于检测。
(3) 组织样品：每10 mg组织加入100 μL 裂解液，冰浴匀浆或超声破碎细胞。4ºC，12,000×g离心5分钟，取上清用于检测。
(4) 细胞培养上清：离心后，直接测定。
2. 标准曲线设置
设置标曲时吸光度法和荧光法二选一即可。对于样品量较少或浓度较低的情况，优先推荐采用荧光检测。吸光度检测时建议使用透明96孔板，荧光检测时建议使用黑色96孔板。
(1) 吸光度检测
取8支EP管，第1支EP管中加入8 µL L-乳酸标准品 (5 mM)和192 µL的检测缓冲液，将标准品稀释为200 μM。其他每管中加入100 μL检测缓冲液。从第1只EP管中吸取100 μL到第2支EP管，混匀，再从第2只EP管中吸取100 μL到第3支EP管中，按此步骤往后依次稀释，最后一支EP管为100 μL检测缓冲液。此时，各个EP管中L-乳酸的浓度依次为200、100、50、25、12.5、6.25、3.125、0 μM。
(2) 荧光检测
取8支EP管，第1支EP管中加入2 µL L-乳酸标准品 (5 mM)和498 µL的检测缓冲液，将标准品稀释为20 μM。其他每管中加入100 μL检测缓冲液。从第1只EP管中吸取100 μL到第2支EP管，混匀，再从第2只EP管中吸取100 μL到第3支EP管，按此步骤往后依次稀释，最后一支EP管为100 μL检测缓冲液。此时，各个EP管中过 L-乳酸的浓度依次为20、10、5、2.5、1.25、0.625、0.313、0 μM。
3. 显色液的配制
根据标准品和样品的数量，按照每个反应50 µL的体积配制显色工作液。具体配制方法参考下表，建议现配现用。
样本数 | 1 | 10 | 20 | 50 |
检测缓冲液 | 44 μL | 440 μL | 880 μL | 2200 μL |
乳酸氧化酶 | 2 μL | 20 μL | 40 μL | 100 μL |
辣根过氧化物酶 | 2 μL | 20 μL | 40 μL | 100 μL |
Amplex Red | 2 μL | 20 μL | 40 μL | 100 μL |
总体积 | 50 μL | 500 μL | 1000 μL | 2500 μL |
注： H₂O₂ 的存在会对检测产生干扰。如果样品含有 H₂O₂ ，需同时设置样品背景对照孔，加入不含乳酸氧化酶的显色液，即配制显色液时2 µL的乳酸氧化酶用检测缓冲液替代。计算时样品孔的读数值需要减去样品背景对照孔的读数。
4. 标准品和样品测定
(1) 取50 μL梯度稀释的标准品和50 μL样本加入到96孔板中。
(2) 各孔加入显色工作液50 µL，混匀，37ºC避光反应30分钟。
(3) 如果使用吸光度检测，测定OD570 nm；如果使用荧光检测，设置激发波长为560 nm，发射波长为590 nm进行荧光强度检测。
5. 结果计算
以L-乳酸标准品的浓度为横坐标，吸光度或荧光强度为纵坐标，建立标准曲线，计算样品中L-乳酸的浓度(A). 如果样品的背景对照信号比较高，样品的信号值应减去样品背景对照的信号值。L-乳酸浓度的计算公式如下：C (μM) = A × n.
注：A为根据标准曲线确定的L-乳酸浓度(μM)；n为样品总稀释倍数。
6. 结果展示
注意事项
1. 酶工作液冰上解冻，使用前后避免室温放置。
2. 样本中L-乳酸的浓度太高时，会出现短暂生成粉色产物然后快速变色现象，因此正式检测前，建议选择2-3个预期差异较大的样本进行预实验。
3. Amplex Red在空气中不太稳定，开启后应尽快使用，且在使用过程中需注意适当避光。
4. 吸光度检测时建议使用透明96孔板，荧光检测时建议使用黑色96孔板。
5. 生化检测试剂普遍具有刺激性、生物毒性等，为了您的安全和健康，请在实验全程做好生物安全防护。
Product Introduction
L‑Lactate is oxidized by lactate oxidase in the presence of oxygen to produce pyruvate and hydrogen peroxide (H₂O₂). In the presence of horseradish peroxidase, Amplex Red reacts with H₂O₂ to generate the strongly red‑fluorescent compound resorufin, which has a maximum excitation wavelength of 571 nm and a maximum emission wavelength of 585 nm, and also exhibits strong visible light absorption at the excitation wavelength. Therefore, this kit can be used for both absorbance and fluorescence detection. The fluorescence intensity or absorbance of resorufin is directly proportional to the L‑lactate concentration in the sample.
Components
L1523443 | Component Name | Appearance | 100 T | Storage | Quantity Per Test |
L1523443A | Lysis Buffer | Liquid | 20 mL | -20°C. | As needed |
L1523443B | Assay Buffer | Liquid | 20 mL | -20°C. | As needed |
L1523443C | Glucose Oxidase | Liquid | 200 μL | -20°C. Store in the dark | 2 μL |
L1523443D | Amplex Red | Liquid | 200 μL | -20°C. Store in the dark | 2 μL |
L1523443E | Horseradish Peroxidase | Liquid | 200 μL | -20°C. Store in the dark | 2 μL |
L1523443F | L-Lactate Standard (5 mM) | Liquid | 500 μL | -20°C. Store in the dark | 8 μL |
Usage Protocol
1. Sample Preparation
1) Serum (plasma) samples: Measure directly.
2) Cell samples: Add 200 μL of lysis buffer per 1 × 10⁶ cells, and lyse on ice for 5-10 minutes or by ultrasonic disruption on ice. Centrifuge at 12,000 × g for 5 minutes at 4 °C, and collect the supernatant for analysis.
3) Tissue samples: Add 100 μL of lysis buffer per 10 mg of tissue, and homogenize or perform ultrasonic disruption on ice. Centrifuge at 12,000 × g for 5 minutes at 4 °C, and collect the supernatant for analysis.
4) Cell culture supernatant: Centrifuge and measure directly.
2. Standard Curve Setup
When setting up the standard curve, choose either the absorbance method or the fluorescence method. For samples with limited volume or low concentration, fluorescence detection is recommended. For absorbance detection, a clear 96‑well plate is recommended; for fluorescence detection, a black 96‑well plate is recommended.
1) Absorbance detection
Take 8 EP tubes. Add 8 µL of L‑Lactate Standard (5 mM) and 192 µL of assay buffer to the first EP tube to dilute the standard to 200 µM. Add 100 µL of assay buffer to each of the remaining tubes. Transfer 100 µL from the first tube to the second tube, mix, then transfer 100 µL from the second to the third, and continue this serial dilution sequentially through the subsequent tubes. The last tube contains 100 µL of assay buffer. The resulting L‑lactate concentrations in each tube are 200, 100, 50, 25, 12.5, 6.25, 3.125, and 0 µM, respectively.
2) Fluorescence detection
Take 8 EP tubes. Add 2 µL of L‑Lactate Standard (5 mM) and 498 µL of Assay Buffer to the first EP tube to dilute the standard to 20 μM. Add 100 μL of Assay Buffer to each of the remaining tubes. Transfer 100 μL from the first tube to the second tube, mix, then transfer 100 μL from the second to the third, and continue this serial dilution through the subsequent tubes. The last tube contains 100 μL of Assay Buffer. At this point, the concentrations of L‑lactate in each tube are 20, 10, 5, 2.5, 1.25, 0.625, 0.313, and 0 μM, respectively.
3. Preparation of Chromogenic Solution
Prepare the Chromogenic Working Solution according to the number of standards and samples, based on a volume of 50 µL per reaction. Refer to the table below for specific preparation methods. It is recommended to prepare the solution fresh immediately before use.
Number of samples | 1 | 10 | 20 | 50 |
Assay Buffer | 44 μL | 440 μL | 880 μL | 2200 μL |
Lactate Oxidase | 2 μL | 20 μL | 40 μL | 100 μL |
Horseradish Peroxidase | 2 μL | 20 μL | 40 μL | 100 μL |
Amplex Red | 2 μL | 20 μL | 40 μL | 100 μL |
Total Volume | 50 μL | 500 μL | 1000 μL | 2500 μL |
Note: The presence of H₂O₂ may interfere with the detection. If the sample contains H₂O₂, a sample background control well must be set up in parallel, using the Chromogenic Solution without Lactate Oxidase (i.e., replace the 2 μL of Lactate Oxidase with Assay Buffer when preparing the Chromogenic Solution). When calculating results, subtract the reading of the sample background control well from that of the sample well.
4. Standard and Sample Assay
1) Add 50 μL of the serially diluted standards and 50 μL of samples to a 96‑well plate.
2) Add 50 μL of Chromogenic Working Solution to each well, mix, and incubate at 37 °C in the dark for 30 minutes.
3) For absorbance detection, measure the OD at 570 nm; for fluorescence detection, measure the fluorescence intensity with excitation at 560 nm and emission at 590 nm.
5. Result Calculation
Plot a standard curve with the L‑Lactate standard concentration on the x‑axis and the absorbance or fluorescence intensity on the y‑axis. Determine the L‑Lactate concentration in the sample (A) from the standard curve. If the background signal of the sample is relatively high, subtract the signal value of the sample background control from that of the sample. Calculate the L‑Lactate concentration using the following formula: C (μM) = A × n.
Note: A is the L‑Lactate concentration (μM) determined from the standard curve; n is the total dilution factor of the sample.
6. Results Presentation
Precautions
1. Thaw the Enzyme Working Solution on ice. Avoid leaving it at room temperature before and after use.
2. When the concentration of L-lactate in the sample is too high, a pink product may be transiently generated and then rapidly change color. Therefore, before formal testing, it is recommended to perform a preliminary experiment using 2-3 samples with expected large differences.
3. Amplex Red is relatively unstable in the air. Use it as soon as possible after opening, and take care to protect it from light during use.
4. For absorbance detection, a clear 96-well plate is recommended; for fluorescence detection, a black 96‑well plate is recommended.
5. Biochemical assay reagents generally have irritancy and biological toxicity. For your safety and health, please take proper biological safety precautions throughout the experiment.
ZJ26F1039954
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