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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。
乳酸是生物体代谢过程中重要的中间产物，与糖代谢、脂类代谢、蛋白质代谢及细胞内能量代谢密切相关，乳酸含量是评估糖元代谢和有氧代谢的重要指标。异常高浓度的乳酸与癌症、糖尿病和乳酸酸中毒等病理状况有关。
本试剂盒检测原理是乳酸脱氢酶催化L-乳酸生成丙酮酸，同时使NAD+还原成NADH和H+, 进一步通过 1-mPMS 的递氢作用与WST-8反应形成黄色可溶性甲臜，测定其在450nm处的吸光度值即可计算样本中L 乳酸的含量。
检测范围：0.03-2 mM
灵敏度： 0.03 mM
适用样本：动植物组织、细胞、细菌、血清（浆）或其他液体
| L1501211 | Component | 48T | 96T | Storage |
| L1501211A | Lactate Assay Buffer | 70 mL | 70 mL×2 | 2-8℃ |
| L1501211B | Lactate Dehydrogenase | 0.7 mL | 1.4 mL | -20℃ |
| L1501211C | Lactate Dehydrogenase Cofactor | 0.5 mL | 1 mL | -20℃ |
| L1501211D | WST-8 | 350 μL | 700 μL | -20℃. Store in the dark. |
| L1501211E | Enhancer | 70 μL | 140 μL | -20℃. Store in the dark. |
| L1501211F | L(+)-Lactate Standard (100 mM) | 50 μL | 100 μL | -20℃ |
请在实验前检查各组分的量。
各组分在规格基础上额外提供 10% 的量用于进行标准曲线制作或预实验。
自备仪器与试剂
| 自备资源类型 | 资源名称 | 备注 |
| 仪器 | 酶标仪 | 能测 450 nm 处的吸光度 |
| 耗材 | 96 孔酶标板 | 普通透明板 |
| 试剂 | PBS (pH 7.4) | 清洗样本用 |
| 其他 | 匀浆器（组织样本）、恒温箱、制冰机、低温离心机、可调节式移液枪及枪头 | 检测量较大时合理使用多通道移液器有助于提高实验效率 |
实验流程
1、试剂准备
| 试剂名称 | 试剂准备 | 注意事项 |
| Lactate Assay Buffer | 即用型；使用前平衡到室温 | 4℃保存 |
| Lactate Dehydrogenase | 即用型； | 实验过程中水上放置；分装保存于-20℃ |
| Lactate Dehydrogenase Cofactor | 即用型； | 实验过程中水上放置；分装保存于-20℃ |
| WST-8 | 即用型； | 实验过程中水上放置；分装保存于-20℃ |
| Enhancer | 即用型； | 实验过程中水上避光放置；分装避光保存于 -20℃ |
| L(+)-Lactate Standard (100 mM) | 使用前平衡到室温 | 100 mM，分装保存于 -20℃ |
2、标准品配制
将 20 μL 100 mM 的标准品加入 980 μL Lactate Assay Buffer 中，配制为 2 mM 的标准品储备液，分装于 -20℃ 保存 6 个月。使用 Lactate Assay Buffer 将 2 mM 标准品储备液依次稀释至终浓度为 1 mM、0.5 mM、0.25 mM、0.125 mM、0.0625 mM、0.03125 mM 的标准品工作液，以 Lactate Assay Buffer 作为空白孔。
| 标准品工作液 | 标准品（μL） | LactateAssayBuffer（μL） | 浓度（mM） |
| 1 | 200μL 2mM | 0 | 2 |
| 2 | 200μL 2mM | 200 | 1 |
| 3 | 200μL 1mM | 200 | 0.5 |
| 4 | 200μL 0.5mM | 200 | 0.25 |
| 5 | 200μL 0.25mM | 200 | 0.125 |
| 6 | 200μL 0.125mM | 200 | 0.0625 |
| 7 | 200μL 0.0625mM | 200 | 0.03125 |
| 空白 | 0 | 200 | 0 |
3、样本制备
注意：推荐使用新鲜样本，如果不立即进行实验，样本可在 -80℃ 保存 1 个月。细胞或组织提取物中的 NADH 或 NADPH 会产生乳酸测定的背景，为了去除 NADH 或 NADPH 背景，同样数量的样品可以在没有乳酸脱氢酶的情况下进行检测，然后从乳酸读数中减去背景读数。内源性乳酸脱氢酶（LDH）可降解乳酸。含有 LDH（如细胞培养液、细胞或组织裂解液等）的样品应通过 10 kDa MW 超滤管（12000 g，4℃ 离心 10 min，具体过滤步骤以说明书为准），以去除所有蛋白质，取滤液进行检测，然后 -80℃ 保存。
3.1 动植物组织：称取约 0.1 g 组织样本，加入 1 mL Lactate Assay Buffer，冰浴匀浆，12000 g，4℃ 离心 5 min，取上清液于新离心管中，置冰上待检测。
3.2 细胞或细菌样本：朱传侠收集 5×10 <sup>6</sup>朱传侠个细胞，用预冷的 PBS 清洗细胞或细菌，800 g 离心 2 min 后弃上清，加入 1 mL Lactate Assay Buffer，冰浴超声波破碎 5 min（功率 20% 或 200 W，超声 3 s，间隔 7 s，重复 30 次），然后 12000 g，4℃ 离心 5 min，取上清液，置冰上待测。
3.3 血浆和血清（其他生物体液）：直接检测。
4、实验步骤
4.1 酶标仪准备：预热 30 min 以上，调节波长到 450 nm。
4.2 工作液的配制：每孔需要 50 μL Working Reagent，为避免损失，按 55 μL 每单孔体系配制：吸取 31 μL Lactate Assay Buffer，8 μL Lactate Dehydrogenase Cofactor，5 μL WST-8，1 μL Enhancer 和 10 μL Lactate Dehydrogenase，混合均匀。Working Reagent 需现配现用。
4.3 检测体系配制：按如下体系在酶标板中进行检测体系配制。标准曲线一般只做 1 次。
| 试剂 | 标准孔（μL） | 测定孔（μL） |
| 样本 | 0 | 50 |
| 标准品工作液 | 50 | 0 |
| Working Reagent | 50 | 50 |
5.1 数据处理
计算ΔA标准=A标准-A空白 ，ΔA测定 = A测定-A空白 。
5.2 标准曲线绘制
以标准品浓度为 y 轴，ΔA标准为 x 轴，绘制标准曲线。将ΔA标准代入方程得到 y 值（mM）。
5.3 样本L-乳酸含量计算
① 按样本质量计算：
L-乳酸 (μmol/g) = y × V样品 ÷ (W × V样总 ÷ V样总 ) × n = y ÷ W × n
② 按细胞或细菌数量计算：
L-乳酸 (μmol/104 )=y×V样品 ÷(500×V样品 ÷V样总 )×n=y÷500×n
③按液体体积计算 ：
L-乳酸 (mM)=y×V样品 ÷V样品 ×n=y×n
④ 按蛋白浓度数量计算：
L-乳酸 (μmol/ mg prot)=y×V样品 ÷(V样品 ×Cpr)×n=y÷Cpr×n
参数说明：
1 mM = 1 mM；
V样总 ：加入样本体积，0.05 mL；
n：样品稀释倍数；
Cpr：样本蛋白质浓度，mg/mL；
W：样品质量，g；
V样品 ：样本提取体积，1 mL；
500：细胞或细菌数量，5×106，以 10 4 为单位。
结果展示用之前的标曲
典型标准曲线：y=2.2613x-0.0531
示例-1: 取50μL鸡血清，按照测定步骤操作，用96孔板进行检测。测得ΔA测定=A测定-A空白=0.435-0.096=0.339， 代入标准曲线，计算y=0.713。按液体体积计算得：乳酸含量 (mM)=y×V样品÷V×n=y×n=0.713×5=3.565mM。
注意事项
1、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。
2、对于组织样本及细胞样本等，可通过测定蛋白浓度来进行样本间结果均一化。推荐使用阿拉丁B665595 BCA蛋白定量试剂盒或R1491648 即用型BCA蛋白定量试剂盒。
3、本试剂盒兼容分光光度计检测，请按照分光光度计检测要求按比例调节检测试剂配制量。
4、建议实验者自行建立标准曲线以提高结果准确度。如未自行建立标准曲线，可参考结果展示部分的典型标准曲线公式进行结果计算。
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， 穿戴实验服、口罩、手套、头套等安全护具，在通风橱、生物安全柜中进行实验。
6、本产品仅供科学研究使用，不适用于临床诊断。
检测得到的样本ΔA测定过高或者过低怎么办？
如果样本ΔA测定>1.0，则说明样本乳酸含量太高，需用Lactate Assay Buffer适当稀释（计算公式中乘以相应稀释倍数）。如果样本的ΔA测定＜0.13，可提高样本量。
Lactic acid is an important intermediate product in the metabolic processes of organisms, closely related to carbohydrate metabolism, lipid metabolism, protein metabolism, and intracellular energy metabolism. Lactic acid concentration is a key indicator for evaluating glycogen metabolism and aerobic metabolism. Abnormally high concentrations of lactic acid are associated with pathological conditions such as cancer, diabetes, and lactic acidosis.
The detection principle of this kit is as follows: Lactate dehydrogenase catalyzes the conversion of L-lactate to pyruvate, simultaneously reducing NAD+ to NADH and H+. Further, through the hydrogen transfer action of 1-mPMS, WST-8 reacts to form a yellow, soluble formazan. The absorbance at 450 nm is measured to calculate the L-lactate content in the sample.
Detection Range: 0.03-2 mM
Sensitivity: 0.03 mM
Applicable Samples: Animal and plant tissues, cells, bacteria, serum (plasma), or other liquids.
| L1501211 | Component | 48T | 96T | Storage |
| L1501211A | Lactate Assay Buffer | 70 mL | 70 mL×2 | 2-8℃ |
| L1501211B | Lactate Dehydrogenase | 0.7 mL | 1.4 mL | -20℃ |
| L1501211C | Lactate Dehydrogenase Cofactor | 0.5 mL | 1 mL | -20℃ |
| L1501211D | WST-8 | 350 μL | 700 μL | -20℃. Store in the dark. |
| L1501211E | Enhancer | 70 μL | 140 μL | -20℃. Store in the dark. |
| L1501211F | L(+)-Lactate Standard (100 mM) | 50 μL | 100 μL | -20℃ |
Please check the quantity of each component before the experiment.
An additional 10% of each component is provided beyond the specified volume for standard curve preparation or preliminary experiments.
User-Provided Instruments and Reagents
| Type | Name | Notes |
| Instrument | Microplate Reader | Capable of measuring absorbance at 450 nm. |
| Consumables | 96-well Microplate | Standard transparent plate. |
| Reagents | PBS (pH 7.4) | For washing samples. |
| Others | Homogenizer (for tissue samples), incubator, ice machine, low-temperature centrifuge, adjustable pipettes and tips | Using a multichannel pipette for large-scale detection can improve efficiency. |
Experimental Procedure
1. Reagent Preparation
| Reagent Name | Reagent Preparation | Precautions |
| Lactate Assay Buffer | Ready-to-use; equilibrate to room temperature before use. | 4℃保存 Store at 4°C. |
| Lactate Dehydrogenase | Ready-to-use; | Keep on ice during the experiment; store aliquots at -20°C. |
| Lactate Dehydrogenase Cofactor | Ready-to-use; | Keep on ice during the experiment; store aliquots at -20°C. |
| WST-8 | Ready-to-use; | Keep on ice during the experiment; store aliquots at -20°C. |
| Enhancer | Ready-to-use; | Keep on ice protected from light during the experiment; store aliquots at -20°C protected from light. |
| L(+)-Lactate Standard (100 mM) | Equilibrate to room temperature before use. | 100 mM, store aliquots at -20°C. |
2. Standard Preparation
Add 20 μL of the 100 mM standard to 980 μL of Lactate Assay Buffer to prepare a 2 mM standard stock solution. Aliquot and store at -20°C for up to 6 months. Dilute the 2 mM standard stock solution sequentially with Lactate Assay Buffer to prepare standard working solutions with final concentrations of 1 mM, 0.5 mM, 0.25 mM, 0.125 mM, 0.0625 mM, and 0.03125 mM. Use Lactate Assay Buffer as the blank.
| Standard Working Solution | Standard (μL) | Lactate Assay Buffer (μL) | Concentration (mM) |
| 1 | 200 μL of 2 mM | 0 | 2 |
| 2 | 200 μL of 2 mM | 200 | 1 |
| 3 | 200 μL of 1 mM | 200 | 0.5 |
| 4 | 200 μL of 0.5 mM | 200 | 0.25 |
| 5 | 200 μL of 0.25 mM | 200 | 0.125 |
| 6 | 200 μL of 0.125 mM | 200 | 0.0625 |
| 7 | 200 μL of 0.0625 mM | 200 | 0.03125 |
| Blank | 0 | 200 | 0 |
3. Sample Preparation
Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 1 month. NADH or NADPH present in cell or tissue extracts can create background for lactate assay. To remove NADH or NADPH background, an equal amount of sample can be assayed without lactate dehydrogenase, and the background reading should be subtracted from the lactate reading. Endogenous lactate dehydrogenase (LDH) can degrade lactate. Samples containing LDH (e.g., cell culture medium, cell or tissue lysates) should be processed using a 10 kDa MW cutoff ultrafiltration tube (centrifuge at 12,000 g, 4°C for 10 min; follow the filter instructions) to remove all proteins. Use the filtrate for detection, then store at -80°C.
3.1 Animal/Plant Tissues: Weigh approximately 0.1 g of tissue sample, add 1 mL of Lactate Assay Buffer, and homogenize on ice. Centrifuge at 12,000 g, 4°C for 5 min. Transfer the supernatant to a new tube and keep on ice for detection.
3.2 Cells or Bacteria: Collect 5×10^6 cells. Wash the cells or bacteria with pre-cooled PBS. Centrifuge at 800 g for 2 min, discard the supernatant. Add 1 mL of Lactate Assay Buffer, and disrupt using an ultrasonic homogenizer on ice for 5 min (power 20% or 200 W, ultrasonic 3 s, interval 7 s, repeat 30 times). Centrifuge at 12,000 g, 4°C for 5 min. Collect the supernatant and keep on ice for detection.
3.3 Plasma and Serum (Other Biological Fluids): Detect directly.
4. Experimental Steps
4.1 Microplate Reader Preparation: Preheat for at least 30 minutes, set wavelength to 450 nm.
4.2 Working Reagent Preparation: 50 μL of Working Reagent is required per well. To avoid loss, prepare for 55 μL per single well system: Pipette 31 μL Lactate Assay Buffer, 8 μL Lactate Dehydrogenase Cofactor, 5 μL WST-8, 1 μL Enhancer, and 10 μL Lactate Dehydrogenase. Mix well. The Working Reagent must be prepared freshly and used immediately.
4.3 Assay System Setup: Set up the detection system in the microplate according to the table below. The standard curve generally needs to be performed only once.
| Reagent | Standard Well (μL) | Test Well (μL) |
| Sample | 0 | 50 |
| Standard Working Solution | 50 | 0 |
| Working Reagent | 50 | 50 |
4.4 Absorbance Measurement: Mix well and incubate at 37°C protected from light for 30 min. Read the absorbance at 450 nm, recorded as Ablank, Astandard, and Atest .
5. Result Calculation
The following provides both the derived formula and the simplified calculation formula, which are completely equivalent.
5.1 Data Processing
Calculate ΔAstandard= Astandard- Ablank, ΔAtest = Atest - Ablank.
5.2 Standard Curve Plotting
Plot the standard curve with standard concentration as the y-axis and ΔAstandard as the x-axis. Substitute ΔAstandard into the equation to obtain the y value (mM).
5.3 Sample L-Lactate Content Calculation
① Calculated based on sample weight:
L-Lactate (μmol/g) = y × Vsample ÷ (W × Vsample ÷ Vtotal) × n = y ÷ W × n
② Calculated based on cell or bacterial count:
L-Lactate (μmol/10⁴ cells) = y × Vsample ÷ (500 × Vsample ÷ Vtotal) × n = y ÷ 500 × n
③ Calculated based on liquid volume:
L-Lactate (mM) = y × Vsample ÷ Vsample × n = y × n
④ Calculated based on protein concentration:
L-Lactate (μmol/mg prot) = y × Vsample ÷ (Vsample × Cpr) × n = y ÷ Cpr × n
Parameter Description:
1 mM = 1 mmol/L;
Vsample : Volume of sample added, 0.05 mL;
n: Sample dilution factor;
Cpr: Sample protein concentration, mg/mL;
W: Sample weight, g;
Vtotal: Total volume of sample extract, 1 mL;
500: Cell or bacterial count, 5×10⁶, converted to units of 10⁴.
Result Presentation Using Previous Standard Curve
Typical Standard Curve: y = 2.2613x - 0.0531
Example-1: 50 μL of chicken serum was taken and processed according to the assay steps using a 96-well plate. The measured ΔAtest = Atest - Ablank= 0.435 - 0.096 = 0.339. Substituting into the standard curve, y = 0.713 mM. Calculated based on liquid volume: Lactate content (mM) = y × n = 0.713 × 5 = 3.565 mM.
Precautions
It is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.
For tissue and cell samples, results can be normalized by measuring the protein concentration.
This kit is compatible with spectrophotometer detection. Adjust the preparation volume of detection reagents proportionally according to the spectrophotometer's requirements.
It is recommended to establish your own standard curve for improved accuracy. If not, you may refer to the typical standard curve formula provided in the results section for calculation.
Biochemical reagents are generally irritating and biologically toxic. For your safety and health, please wear appropriate personal protective equipment (lab coat, mask, gloves, hair cap, etc.) throughout the experiment and perform experiments in a fume hood or biosafety cabinet.
This product is for scientific research use only. Not intended for clinical diagnosis.
Frequently Asked Questions
What should I do if the sample ΔAtest is too high or too low?
If the sample ΔAtest is >1.0, the lactate content in the sample is too high. Dilute the sample appropriately with Lactate Assay Buffer (multiply by the dilution factor in the calculation). If the sample ΔAtest is <0.13, increase the sample amount.
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