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乳酸脱氢酶细胞毒性检测试剂盒(LDH Cytotoxicity Assay Kit)，也称乳酸脱氢酶检测试剂盒(LDH Assay Kit) 或乳酸脱氢酶释放检测试剂盒(LDH Release Assay Kit)，是一种基于diaphorase催化的INT显色反应。通过检测吸光度检测细胞毒性时释放的乳酸脱氢酶活性或检测其它样品中的乳酸脱氢酶活性的试剂盒。
本试剂盒可以用于常规的乳酸脱氢酶活性的检测，更常用于以LDH 释放为指标的细胞毒性检测。同时，基于细胞总乳酸脱氢酶活性的检测，本试剂盒也可以用于检测细胞增殖和细胞毒性检测。
细胞凋亡或坏死而造成的细胞膜结构的破坏会导致细胞浆内的酶释放到培养液里，其中包括酶活性较为稳定的乳酸脱氢酶(lactate dehydrogenase, LDH)。通过检测从质膜破裂的细胞中释放到培养液中的LDH的活性，就可以实现对细胞毒性的定量分析。LDH 释放被看做细胞膜完整性的重要指标，并被广泛用于细胞毒性检测。LDH 释放被认为是以前使用放射性的⁵¹Cr标记细胞，随后通过⁵¹Cr释放进行细胞膜完整性检测的安全有效的替代方法。
本试剂盒的基本原理是，在乳酸脱氢酶的作用下，NAD+被还原生成NADH，NADH和INT (2-p-iodophenyl-3-nitrophenyl tetrazolium chloride)被硫辛酰胺脱氢酶(diaphorase)催化反应生成NAD+和强生色物甲臜(formazan)，在490nm波⻓下产生吸收峰，从而通过比色来定量检测细胞培养液、细胞裂解液等样品中乳酸脱氢酶的活性。吸光度与乳酸脱氢酶活性成线性正相关。该酶联反应原理的示意图如下：
产品组分及储存条件：
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注意事项：
1. 冻会使样品中部分乳酸脱氢酶失活，4°C可放置2-3天。建议样品准备好后尽量当天完成测定。
2. 如果检测细胞培养液中的乳酸脱氢酶，由于血清含有乳酸脱氢酶，建议血清的使用浓度不要超过1%，并最好使用热灭活血清。如果一定需要使用10%血清，在检测时一定要设置没有细胞，但加入了相同体积培养液的对照孔，以用于消除背景。
3. 细胞过度生⻓、密度过高、离心速度过大、培养箱内外温差过大，都会造成细胞释放乳酸脱氢酶增加。4. 如果希望进行乳酸脱氢酶活性的绝对定量，用户需自备乳酸脱氢酶标准品。
5. 本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于⻝品或药品，不得存放于普通住宅内。
6. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
使用方法：
1. 样品的准备：
方法一：LDH释放检测
① 根据细胞的大小和生⻓速度将适量细胞接种到96孔细胞培养板中，使待检测时细胞密度不超过80-90%。
② 吸去培养液，用PBS液洗涤一次。换新鲜培养液 (推荐使用含1%血清的低血清培养液或适当的无血清培养液)，将各培养孔分成如下几组：包括无细胞的培养液孔 (背景空白对照孔)，未经药物处理的对照细胞孔 (样品对照孔)，未经药物处理的用于后续裂解的细胞孔 (样品最大酶活性对照孔)，以及药物处理的细胞孔(药物处理样品孔)，并做好标记。按照实验需要给予适当药物处理 (如加入0-10μl左右特定的药物刺激，可设置不同浓度，不同处理时间，对照孔中需加入适当的药物溶剂对照)，继续按常规培养。到预定的检测时间点前1小时，从细胞培养箱里取出细胞培养板，在“样品最大酶活性对照孔”中加入试剂盒提供的LDH释放试剂，加入量为原有培养液体积的10%。加入LDH释放试剂后，反复吹打数次混匀，然后继续在细胞培养箱中孵育。
③ 到达预定时间后，将细胞培养板用多孔板离心机400g离心5min。分别取各孔的上清液120μl，加入到一新的96孔板相应孔中，随即进行样品测定。
方法二：细胞内总LDH的检测
① 细胞毒性检测：根据细胞的大小和生⻓速度将适量细胞接种到96孔细胞培养孔板中，使待检测时细胞密度不超过80-90%。加入不同药物进行处理，并设置适当对照。药物刺激完毕后，将细胞培养板用多孔板离心机400g离心5min。尽量吸除上清，加入150μl用PBS稀释了10倍的试剂盒提供的LDH释放试剂 (10 体积PBS中加入1体积LDH释放试剂并混匀)，适当摇晃培养板混匀，然后继续在细胞培养箱中孵育1小时。随后将细胞培养板用多孔板离心机400g离心5min。分别取各孔的上清液120μl，加入到一新的96孔板相应孔中，随即进行样品测定。
② 细胞增殖检测：根据细胞的大小和生⻓速度将适量细胞接种到96孔细胞培养孔板中，使促进细胞增殖的药物刺激后细胞不超过80-90%满为宜。使用不同的药物刺激细胞，并设置适当对照。药物刺激完毕后，将细胞培养板用多孔板离心机400g离心5min。尽量吸除上清，加入150μl用PBS稀释了10倍的试剂盒提供的LDH释放试剂 (10体积PBS中加入1体积LDH释放试剂并混匀)，适当摇晃混匀胞，然后继续在细培养箱中孵育1小时。随后将细胞培养板用多孔板离心机400g离心5min。分别取各孔上清液120μl，加入到一新的96孔板相应孔中，随即进行样品测定。
注：LDH释放检测更加常用一些，细胞内总LDH检测通常可以使用MTT、WST-1或CCK-8等方法替代。
2.试剂盒的准备工作：
① INT溶液 (1×) 的配置：根据所需的INT溶液 (1×) 的量，取适量INT溶液 (10×) 用INT稀释液稀释至1×。
例如，取20μl INT溶液 (10×)，加入180μl INT稀释液，混匀后即配置为200μl INT溶液 (1×)。INT溶液 (1×)宜现配现用，配置后4°C保存可于当天使用，不宜配置后冻存。
② LDH检测工作液的配制：根据待测定的样品数 (含对照)，参考下表在临检测前新鲜配制适量的检测工作液。
注意：LDH检测工作液必须现配现用，配制和使用过程中均要注意适当避光。
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③ (选做) 如果希望进行LDH酶活性的绝对定量，需自备LDH标准品，并新鲜配制不同浓度LDH标准品，如10mU/ml、5mU/ml、2.5mU/ml、1.25mU/ml、0.65mU/ml、0mU/ml。
3. 样品测定：
① 各孔分别加入60μl LDH检测工作液。
② 混匀，室温 (约25°C) 避光孵育30min (可用铝箔包裹后置于水平摇床或侧摆摇床上缓慢摇动)。然后在490nm处测定吸光度。使用600nm或大于600nm的任一波⻓作为参考波⻓进行双波⻓测定。
③ 计算 (测得的各组吸光度均应减去背景空白对照孔吸光度)。
④ 细胞毒性或死亡率 (%) = (处理样品吸光度－样品对照孔吸光度) / (细胞最大酶活性的吸光度－样品对照孔吸光度)×100
⑤ 可绘制细胞毒性曲线：纵坐标为实际吸光度，横坐标为药物浓度；据此可计算该药物作用特定时间的半致死剂量LD50。
附录1：
可同时测定一已知浓度的LDH酶标准品对应的吸光度值，参考以下公式粗略计算出样品中LDH酶活力：
待测样品中LDH活力单位 (mU/ml)＝(样品孔吸光度－背景空白对照孔吸光度) / (标准管吸光度－标准空白管吸光度)×标准品浓度(mU/ml)；
根据计算结果可以比较不同样品处理组间有无统计学差异等。
附录2：
若需准确计算出LDH酶活性的绝对活性，可通过一系列LDH标准品及相应测得的吸光度值绘制标准曲线，通过标准曲线相应公式计算出样品中LDH的酶活性。
各孔数值减去空白对照后，以检测的吸光度(OD490)为纵坐标，LDH酶活力(mU)为横坐标，绘制LDH标准曲线。同时计算出该趋势线的公式。
图1. 典型乳酸脱氢酶 (LDH) 标准曲线
A₄₉₀ₙₘ＝k×LDH酶活力单位(mU)＋b，通过Excel等软件计算出趋势线的斜率k和截距b。
根据上述公式计算样品中LDH活力。
样品实际吸光度(OD₄₉₀)＝样品孔测得的吸光度－背景空白对照孔吸光度。
检测体系中LDH酶活力单位(mU)=(OD₄₉₀-b)/k。
样品中LDH酶活力(mU/ml)＝检测体系中LDH酶活力单位(mU )/检测样品体积。
图2. LDH 试剂盒用于培养的人星形胶质细胞 (Has)；实心柱为经 Triton X-100 处理组 (阳性对照)，空心柱为未处理组 (阴性对照)
LDH Cytotoxicity Assay Kit, also referred to as LDH Assay Kit or LDH Release Assay Kit, relies on an INT colorimetric reaction catalyzed by diaphorase. A kit for measuring the activity of lactate dehydrogenase released during cytotoxicity via absorbance detection, or detecting lactate dehydrogenase activity in other samples.
This kit can be used for routine detection of lactate dehydrogenase activity, and is more commonly applied to cytotoxicity assays using LDH release as an indicator. Meanwhile, based on the measurement of total cellular lactate dehydrogenase activity, the kit is also suitable for cell proliferation and cytotoxicity detection.
Disruption of cell membrane structure caused by cell apoptosis or necrosis leads to the release of cytoplasmic enzymes into the culture medium, including lactate dehydrogenase (LDH) with relatively stable enzymatic activity. Quantitative analysis of cytotoxicity can be achieved by measuring the activity of LDH released into the culture medium from cells with ruptured plasma membranes. LDH release serves as a key marker of plasma membrane integrity and is widely adopted in cytotoxicity testing. LDH release assay is recognized as a safe and effective alternative to the traditional radioactive ⁵¹Cr release assay, in which cells are labeled with radioactive ⁵¹Cr to evaluate plasma membrane integrity.
The core principle of this kit is described as follows: LDH catalyzes the reduction of NAD⁺ to NADH. Subsequently, NADH reacts with INT (2-p-iodophenyl-3-nitrophenyl tetrazolium chloride) under the catalysis of diaphorase to regenerate NAD⁺ and form intensely colored formazan, which exhibits an absorption peak at 490 nm. The LDH activity in samples such as cell culture supernatants and cell lysates can therefore be quantitatively determined by colorimetric measurement. Absorbance shows a positive linear correlation with LDH activity. A schematic diagram illustrating this coupled enzymatic reaction is presented below:
Product Components and Storage Conditions:
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Precautions:
1. Freezing will inactivate part of lactate dehydrogenase in samples. Samples can be stored at 4°C for 2–3 days. It is recommended to complete the assay on the same day after sample preparation.
2. When detecting LDH in cell culture medium, serum contains endogenous LDH. Therefore, the serum concentration should not exceed 1%, and heat-inactivated serum is preferred. If 10% serum is mandatory, control wells containing the same volume of culture medium without cells must be set up to eliminate background interference during detection.
3. Excessive cell overgrowth, ultra-high cell density, excessive centrifugal force, and large temperature differences inside and outside the incubator will lead to elevated LDH release from cells.
4. For absolute quantification of LDH activity, users need to prepare LDH standard by themselves.
5. This product is for scientific research use only by professional researchers. It shall not be used for clinical diagnosis or treatment, food or pharmaceutical production, nor stored in residential premises.
6. For personal safety and health, wear a lab coat and disposable gloves during all operations.
Operating Instructions:
1. Sample Preparation:
Method 1: LDH Release Assay
① Inoculate appropriate amounts of cells into a 96-well cell culture plate according to cell size and growth rate, so that the cell confluency does not exceed 80–90% at detection.
② Aspirate culture medium and wash cells once with PBS. Replace with fresh medium (low-serum medium containing 1% serum or suitable serum-free medium is recommended). Divide all wells into the following groups with clear labeling: Medium-only wells (background blank control wells, no cells). Untreated cell wells (sample spontaneous release control wells). Untreated cell wells reserved for complete lysis (maximum LDH activity control wells). Drug-treated cell wells (experimental sample wells)
Treat cells with test agents as required by your experiment (add 0–10 μL of specific stimulants per well; multiple concentrations and treatment durations can be set). Add an equal volume of drug solvent to control wells as solvent blank, then incubate under standard culture conditions.
One hour before the scheduled detection time, remove the plate from the incubator. Add the kit-supplied LDH Release Reagent to maximum LDH activity control wells at a volume equal to 10% of the existing medium. Mix thoroughly by repeated pipetting, then return the plate to the incubator for continued incubation.
③ Upon reaching the preset incubation time, centrifuge the plate at 400 × g for 5 min using a microplate centrifuge. Transfer 120 μL supernatant from each well to corresponding wells of a new 96-well plate, and proceed with sample measurement immediately.
Method 2: Intracellular Total LDH Assay
① Cytotoxicity Detection:
Seed appropriate cells into a 96-well culture plate based on cell size and proliferation speed, with cell confluency controlled below 80–90% at detection. Treat cells with different drugs and set up corresponding control groups. After drug treatment, centrifuge the plate at 400 × g for 5 min with a microplate centrifuge. Aspirate supernatant as completely as possible, then add 150 μL 10-fold diluted LDH Release Reagent (prepared by mixing 1 volume of kit LDH Release Reagent with 10 volumes of PBS). Gently shake the plate to homogenize, then incubate in the cell incubator for 1 h. Centrifuge the plate again at 400 × g for 5 min. Transfer 120 μL supernatant from each well to a new 96-well plate, and test samples right away.
② Cell Proliferation Detection:
Inoculate suitable cells into a 96-well culture plate according to cell characteristics; ensure cell density does not exceed 80–90% after stimulation with proliferation-promoting agents. Stimulate cells with different compounds and set matched controls. Complete drug treatment, then centrifuge the plate at 400 × g for 5 min. Remove all supernatant, add 150 μL 10-fold diluted LDH Release Reagent (1 part LDH Release Reagent + 10 parts PBS). Shake the plate gently for uniform mixing, and incubate in the cell incubator for 1 h. Centrifuge the plate at 400 × g for 5 min, transfer 120 μL supernatant per well to a new 96-well plate, and conduct sample detection without delay.
Note: The LDH release assay is more widely applied. The intracellular total LDH measurement can generally be substituted by MTT, WST-1 or CCK-8 assays.
2. Kit Preparation
2. Kit Preparation
① Preparation of 1× INT Solution:
According to the required volume of 1× INT Solution, dilute an appropriate amount of 10× INT Solution with INT Diluent to 1× working concentration.
Example: Take 20 μL of 10× INT Solution and add 180 μL INT Diluent, mix well to prepare 200 μL 1× INT Solution.
1× INT Solution should be freshly prepared before use. It can be stored at 4 °C and used on the same day; freezing after preparation is not recommended.
② Preparation of LDH Assay Working Solution:
Prepare an appropriate volume of fresh assay working solution right before testing according to the number of samples to be detected (including controls), as shown in the table below.
Note: LDH Assay Working Solution must be prepared freshly before use. Avoid excessive light exposure during preparation and operation.
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③ (Optional) For absolute quantification of LDH activity, prepare LDH standard solutions of serial concentrations with self-supplied LDH standard, such as 10 mU/mL, 5 mU/mL, 2.5 mU/mL, 1.25 mU/mL, 0.65 mU/mL and 0 mU/mL.
3. Sample Detection
① Add 60 μL LDH Assay Working Solution to each well respectively.
② Mix thoroughly, incubate at room temperature (approximately 25 °C) in the dark for 30 min (cover the plate with aluminum foil and shake gently on a horizontal or orbital shaker). Afterwards, measure absorbance at 490 nm. Perform dual-wavelength measurement with 600 nm or any wavelength above 600 nm as the reference wavelength.
③ Calculation: Subtract the absorbance of blank background control wells from the measured absorbance of all groups.
④ Cytotoxicity / Cell Death Rate (%) = [(Absorbance of treated sample − Absorbance of sample control well) ÷ (Absorbance of maximum cellular LDH activity − Absorbance of sample control well)] × 100
⑤ Cytotoxicity curve plotting is available: plot corrected absorbance on the vertical axis and drug concentration on the horizontal axis. The half-lethal dose (LD50) of the drug at a specific treatment time can be calculated based on this curve.
Appendix 1:
The absorbance of an LDH standard with known concentration can be measured in parallel. The following formula can be used for rough calculation of LDH activity in samples:
LDH activity of test sample (mU/mL) = [(Absorbance of sample well − Absorbance of background blank well) ÷ (Absorbance of standard well − Absorbance of standard blank well)] × Standard concentration (mU/mL)
Statistical differences between different treatment groups can be analyzed based on the calculated activity values.
Appendix 2:
For accurate quantification of absolute LDH enzymatic activity, generate a standard curve using a serial dilution of LDH standards and their corresponding absorbance readings, then calculate sample LDH activity via the standard curve equation.
After subtracting blank absorbance from all well readings, plot the LDH standard curve with measured absorbance (OD490) on the Y-axis and LDH activity (mU) on the X-axis, and obtain the linear regression equation of the trendline.
Fig1. A Typical LDH standard curve
A₄₉₀ₙₘ = k × LDH activity (mU) + b. The slope k and intercept b of the trendline can be calculated via software such as Excel.
Calculate the LDH activity in the sample using the above formula.
Corrected absorbance of sample (OD₄₉₀) = Absorbance measured in sample wells − Absorbance of background blank control wells.
LDH activity in the reaction system (mU) = (OD₄₉₀ − b) / k.
LDH activity of sample (mU/mL) = LDH activity in the reaction system (mU) / Volume of sample added to the reaction system.
Fig.2 LDH kit is applied to Human Astrocytes(Has)cultured with (Solid bar, Positive control)and without (Open bar, Negative control)Triton X-10
| L1525373 | 物理外观 | 组件 | 100T | 储存 |
| L1525373A | 液体 | LDH释放试剂 | 7.5 ml | 2-8℃. Store in the dark. |
| L1525373B | 液体 | 乳酸溶液 | 10 ml | 2-8℃. Store in the dark. |
| L1525373C | 液体 | 酶溶液 | 5 ml × 2 | 2-8℃. Store in the dark. |
| L1525373D | 液体 | INT溶液 (10×) | 1 ml | 2-8℃. Store in the dark. |
| L1525373E | 液体 | INT稀释液 | 10 ml | 2-8℃. Store in the dark. |
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