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三磷酸腺苷（ATP）是细胞代谢的化学能量来源，常被称作细胞的 “能量货币”。ATP 在活细胞内通过光合作用与细胞呼吸过程生成，并参与各类生命活动的能量消耗，包括生物合成反应、细胞运动及细胞分裂等生理过程。ATP 是反映细胞活性的核心指标，目前已广泛应用于科研与药物研发领域，用于评估细胞活力及细胞毒性。通常细胞在凋亡、坏死或处于一些毒性状态下，ATP水平会下降；而高葡萄糖刺激等可上调某些细胞的细胞内ATP水平。
本试剂盒根据萤火虫萤光素酶(firefly luciferase，也称荧光素酶)催化萤光素产生萤光时需要ATP提供能量研制而成。当萤火虫萤光素酶和萤光素都过量时，在一定的浓度范围内萤光的产生和ATP的浓度成正比。这样就可以高灵敏地检测溶液中的ATP浓度。
注意事项：
1. 试剂盒仅供研究使用，如将其用于临床诊断或任何其他用途，不得用于食品或药品，不得存放于普通住宅内。
2. 实验前请仔细阅读说明书并调整好仪器，严格按照说明书进行实验。
3. 实验中请穿着实验服并戴乳胶手套做好防护工作。
4. ATP检测试剂中含有萤光素酶，反复冻融会导致其逐渐失活。为取得良好的使用效果，第一次解冻后可适当分装保存，但需注意分装的容器不能ATP污染。ATP检测试剂稀释成ATP检测工作液后，最好一次用完，不宜冻存后再使用。
5. ATP，特别是裂解后样品中的ATP在室温不太稳定，需在4ºC或冰上操作。
6. 本试剂盒需使用luminometer，即化学发光仪(检测萤光素酶报告基因时所用的仪器)。如果没有luminometer，也可以使用液闪仪。液闪仪的测定效果取决于液闪仪的检测灵敏度和检测精度。
7. 使用可检测化学发光的多功能酶标仪时，推荐使用孔和孔之间不透光的96孔白板或黑板。如使用普通的透明96孔板，须特别注意在检测孔之间设置间隔孔，以减少邻近孔之间的相互干扰。
8. 每次实验均需同步运行标准品。工作液的荧光本底值会随时间逐渐升高，进而降低检测灵敏度。
9. 由于荧光素-荧光素酶反应灵敏度极高，需避免外源生物源 ATP 污染，例如细菌、指纹等带来的污染。建议使用的超纯水、枪头、离心管、耗材无 ATP 污染。
10. 荧光素、发光反应液对光敏感，光照会加速试剂失活、背景升高，建议实验操作时全程避光。
11. 含萤火虫荧光素酶的溶液请轻柔混匀 (如颠倒混匀)，禁止涡旋振荡，以防酶蛋白变性。
12. 砷酸盐类化合物会抑制本反应体系。此外，样品中的高盐浓度通常会对萤光素酶产生抑制效应，并降低灵敏度。
使用方法
1. 试剂准备
额外所需材料：
(1) 自备试剂：PBS
(2) 所需仪器：多功能酶标仪 (具有检测化学发光检测仪功能)。
(3) 其它：不透光的96孔白板或黑板、低温离心机、匀浆器 (组织样本)。
2. 溶液准备
(1) 1×ATP检测裂解液配制：按照 4:1的比例用去离子水将5×ATP检测裂解液稀释待用。例如：1 mL 5×ATP检测裂解液加入4 mL 去离子水，混匀后即为1×ATP检测裂解液。
(2) 辅因子：即用型；使用前将溶液分装为10份，每份 10 μL，置于≤ -20℃冻保存。已解冻的分装液需置于冰上或 4℃环境暂存，直至使用。
(3) 1×ATP检测缓冲液液配制: 按照 9:1的比例用去离子水将10×ATP检测缓冲液稀释待用。例如：1 mL 10×ATP检测液加入9 mL 去离子水，混匀后即为1×ATP检测缓冲液。
(4) D-荧光素：即用型。临用前分装为10份，置于≤−20℃避光冻保存，直至使用。
(5) 根据待测样品数参考下表配制适当量的ATP检测工作液，表中试剂按比例混合后即为ATP检测工作液。ATP 检测工作液配制完成后，若无法立即使用，需置于避光条件下保存。
注：用不完的试剂分装后避光 -20℃保存，避免反复冻融。 |
1. 样品准备 (注意：样品裂解需在4ºC或冰上操作)
(1) 对于贴壁细胞：
吸除培养液，按照6孔板每孔加入200 μL裂解液的比例 (即相当于细胞培养液量2 mL的1/10)加入裂解液，裂解细胞。裂解细胞时为了裂解充分，可以使用移液器进行反复吹打或晃动培养板使裂解液充分接触并裂解细胞。通常细胞在接触裂解液后会立即裂解。裂解后4ºC 12000×g离心5分钟，取上清，用于后续的测定。
(2) 对于悬浮细胞：
用离心管离心沉淀细胞，弃上清，轻轻弹散细胞，按照6孔板每孔的细胞量加入200 μL裂解液的比例加入裂解液，裂解细胞。裂解细胞时为了裂解充分可以弹击离心管管底或适当Vortex使裂解液充分接触并裂解细胞。通常细胞在接触裂解液后会立即裂解。裂解后4ºC 12000×g离心5分钟，取上清，用于后续的测定。
(3) 对于组织样品:
按照每20 mg组织加入约100-200 μL裂解液的比例加入裂解液，然后用玻璃匀浆器或其它匀浆设备进行匀浆。充分匀浆可以确保组织被完全裂解。裂解后4ºC 12000×g离心5分钟，取上清，用于后续的测定。
2. 标准曲线测定的准备
冰浴上融解待用试剂，把ATP标准溶液用ATP检测裂解液稀释成适当的浓度梯度。具体的浓度需根据样品中ATP的浓度而定。初次检测可以检测 0.01、0.03、0.1、0.3、1、3和10 µM这几个浓度，在后续实验中，可以根据样品中ATP的浓度对标准品的浓度范围进行适当调整。
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1. ATP浓度的测定
(1) 加100 μLATP检测工作液到检测孔或检测管内；
(2) 在检测孔或检测管内加上20 μL样品或标准品，混匀，37°孵育15-20分钟。待反应完成后，用多功能酶标仪测定RLU。
注1：样品的体积可以自行在10-100 µL范围内调节。如果样品中的ATP浓度比较低则可以加入100 µL样品，如果样品中ATP浓度比较高则可以加入较小体积的样品，同时标准品也需要使用相同的体积。如果样品中ATP的浓度特别高，可以用ATP检测裂解液稀释样品后再测定。本试剂盒在加入10-100 µL标准品时，在0.1nM-10 µM的浓度范围内有很好的线性关系。
注2： 对于高浓度 ATP 样品，可适当延长孵育时间 (不超过 30 分钟)，确保反应进入稳定平台期后再读数，以获得最佳线性相关性; 对于低浓度 ATP 样品，在标准孵育条件下信号上升较快，通常在 3-5 分钟即可达到可检测的稳定信号，可提前读数以提高实验效率。
(3) 根据标准曲线计算出样品中ATP的浓度。
Adenosine triphosphate (ATP) is the chemical energy source for cellular metabolism and is commonly known as the "energy currency" of cells. ATP is produced in living cells through photosynthesis and cellular respiration, and provides energy for a variety of vital life processes, including biosynthesis, cell motility, and cell division. As a core indicator reflecting cellular activity, ATP is widely used in scientific research and drug development to evaluate cell viability and cytotoxicity. Typically, intracellular ATP levels decline during cell apoptosis, necrosis, or exposure to toxic conditions; by contrast, stimuli such as high glucose can upregulate intracellular ATP content in certain cell types.
This kit is developed based on the principle that firefly luciferase requires ATP to provide energy for catalyzing the luminescent reaction of D-luciferin. When firefly luciferase and luciferin are present in excess, the luminescence intensity is positively correlated with ATP concentration within a certain linear range, enabling highly sensitive detection of ATP levels in samples.
A1508155 | Components | Appearance | 100T | 10×100T | Storage | Quantity Per Test |
A1508155A | 5×ATP Detection Lysis Buffer | Colorless clear liquid | 20 mL | 10×20 mL | -20°C | On request |
A1508155B | 10×ATP Assay Buffer | Colorless clear liquid | 10 mL | 10×10 mL | -20°C | 100 µL |
A1508155C | D-luciferin | Yellow liquid | 0.1 mL | 10×0.1 mL | -20°C, Protect from light | On request |
A1508155D | Luciferase | Colorless clear liquid | 0.2 mL | 10×0.2 mL | -20°C | On request |
A1508155E | Cofactor | Colorless clear liquid | 0.1 mL | 10×0.1 mL | -20°C | On request |
A1508155F | ATP Standard (500µM) | Colorless clear liquid | 0.1 mL | 10×0.1 mL | -20°C | On request |
Precautions
1. This kit is for research use only. It shall not be used for clinical diagnosis or any other purposes. It is prohibited for food or pharmaceutical applications, and the kit shall not be stored in residential premises.
2. Read the user manual carefully and calibrate all instruments before the experiment. Perform the assay strictly in accordance with the instructions.
3. Wear a lab coat and latex gloves for personal protection during the experiment.
4. The ATP detection reagent contains firefly luciferase, which will gradually lose activity upon repeated freeze-thaw cycles. For optimal performance, aliquot the reagent properly after the first thawing; ensure all aliquot containers are free of ATP contamination. Once diluted into working solution, the ATP detection reagent is recommended for single use only and should not be refrozen for subsequent use.
5. ATP, especially ATP in lysed samples, is unstable at room temperature. All operations should be performed at 4°C or on ice.
6. A luminometer (the instrument commonly used for firefly luciferase reporter gene detection) is required for this assay. A liquid scintillation counter may be used as an alternative; its detection performance depends on the instrument’s sensitivity and precision.
7. When using a multifunctional microplate reader with chemiluminescence detection function, use white or black 96-well plates with light-tight gaps between wells. If standard transparent 96-well plates are adopted, arrange blank spacing wells between sample wells to eliminate cross-well interference.
8. Run standard samples in parallel with each experiment. The background luminescence of working solution increases over time, which will reduce assay sensitivity.
9. The luciferin-luciferase reaction is extremely sensitive; avoid exogenous biological ATP contamination derived from bacteria, fingerprints and other biological sources. Use ATP-free ultrapure water, pipette tips, centrifuge tubes and other consumables throughout the experiment.
10. Luciferin and luminescent reaction buffer are light-sensitive. Light exposure accelerates reagent inactivation and elevates background signals. Keep all operations protected from light.
11. Mix solutions containing firefly luciferase gently by inverting the tube. Do not vortex, as vigorous shaking may cause enzyme denaturation.
12. Arsenate compounds can inhibit this reaction system. In addition, high salt concentration in samples commonly suppresses luciferase activity and reduces detection sensitivity.
Procedure (Unless otherwise specified, all procedures should be performed at 4°C)
1. Reagent Preparation
Additional materials required:
(1) Self-provided reagent: PBS
(2) Required instrument: Multifunctional microplate reader (equipped with chemiluminescence detection function)
(3) Others: Opaque white or black 96-well plate, low-speed refrigerated centrifuge, homogenizer (for tissue samples)
2. Solution Preparation
(1) Preparation of 1×ATP Lysis Buffer: Dilute the 5×ATP Lysis Buffer with deionized water at a ratio of 4:1 for later use. Example: Add 4 mL of deionized water to 1 mL of 5× ATP Lysis Buffer and mix well to prepare 1×ATP Lysis Buffer.
(2) Cofactor: Ready-to-use. Aliquot the solution into 10 equal portions of 10 μL each before use, and store frozen at ≤ -20°C. Thawed aliquots should be kept on ice or at 4°C until use.
(3) Preparation of 1×ATP Assay Buffer: Dilute the 10× ATP Assay Buffer with deionized water at a ratio of 9:1 for later use. Example: Add 9 mL of deionized water to 1 mL of 10× ATP Assay Buffer and mix well to prepare 1× ATP Assay Buffer.
(4) D-luciferin: Ready-to-use. Aliquot into 10 portions immediately before use, store frozen at ≤ -20°C and protected from light until needed.
(5) Prepare an appropriate volume of ATP Working Solution according to the number of test samples with reference to the table below. Mix the related reagents in the specified proportion to obtain the final ATP Working Solution. If the prepared working solution cannot be used immediately, store it under light‑protected conditions.
Note: Unused reagents should be aliquoted and stored at -20°C protected from light to avoid repeated freeze-thaw cycles. |
3. Sample Preparation (Note: Perform all lysis steps at 4°C or on ice)
(1) Adherent cells:
Aspirate the culture medium. Add lysis buffer at a ratio of 200 μL per well of a 6-well plate (equivalent to 1/10 of the 2 mL culture medium volume). To ensure complete lysis, repeatedly pipette the lysate or rock the plate to ensure the lysis buffer fully contacts the cells. Cells typically lyse immediately upon contact with the lysis buffer. After lysis, centrifuge at 12,000×g for 5 minutes at 4°C, then collect the supernatant for subsequent assays.
(2) Suspension cells:
Pellet the cells by centrifugation, discard the supernatant, and gently resuspend the cell pellet. Add lysis buffer at a ratio of 200 μL per well-equivalent cell count (as in a 6-well plate). To ensure complete lysis, tap the bottom of the centrifuge tube or vortex briefly to ensure the lysis buffer fully contacts the cells. Cells typically lyse immediately upon contact with the lysis buffer. After lysis, centrifuge at 12,000×g for 5 minutes at 4°C, then collect the supernatant for subsequent assays.
(3) Tissue samples:
Add lysis buffer at a ratio of approximately 100-200 μL per 20 mg of tissue, then homogenize using a glass homogenizer or other homogenization equipment. Thorough homogenization ensures complete tissue lysis. After lysis, centrifuge at 12,000×g for 5 minutes at 4°C, then collect the supernatant for subsequent assays.
4. Preparation for Standard Curve Assay
Thaw all reagents on ice. Dilute the ATP standard solution with ATP Lysis Buffer to prepare appropriate concentration gradients. The exact concentrations should be determined based on the expected ATP concentration in the samples. For initial testing, concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 µM are recommended. In subsequent experiments, the range of standard concentrations can be adjusted according to the ATP levels in the samples.
Tube ID | Lysis Buffer Volume (μL) | ATP Standard Solution Volume | Final Concentration (μM) |
A | 98 | 2 μL from 0.5 mM ATP standard | 10 |
B | 70 | 30 μL from Tube A | 3 |
C | 90 | 10 μL from Tube A | 1 |
D | 90 | 10 μL from Tube B | 0.3 |
E | 90 | 10 μL from Tube C | 0.1 |
F | 90 | 10 μL from Tube D | 0.03 |
G | 90 | 10 μL from Tube E | 0.01 |
5. ATP Concentration Assay
(1) Add 100 μL of ATP Working Solution to each assay well or tube.
(2) Add 20 μL of sample or standard to each well/tube, mix gently, and incubate at 37°C for 15–20 minutes. After the reaction is complete, measure the RLU (Relative Light Unit) using a multifunctional microplate reader.
Notes1: The sample volume can be adjusted within the range of 10-100 µL as needed. For samples with low ATP levels, use 100 µL of sample; for high ATP levels, use a smaller volume of sample. The same volume must be used for standards and samples. If the ATP concentration in the sample is extremely high, dilute the sample with ATP Lysis Buffer prior to measurement. When using 10-100 µL of standard, the kit exhibits excellent linearity in the range of 0.1 nM to 10 µM.
Notes2: For high-concentration ATP samples, the incubation time may be appropriately extended (no more than 30 minutes) to ensure the reaction reaches a stable plateau before reading and achieve optimal linear correlation; For low-concentration ATP samples, the luminescent signal increases rapidly under standard incubation conditions. A stable and detectable signal is generally achieved within 3-5 minutes, and the measurement can be taken in advance to improve experimental efficiency.
(3) Calculate the ATP concentration in the samples based on the standard curve.
A1508155 | Components | Appearance | 100T | 10×100T | Storage | Quantity Per Test |
A1508155A | 5×ATP Detection Lysis Buffer | Colorless clear liquid | 20 mL | 10×20 mL | -20°C | On request |
A1508155B | 10×ATP Assay Buffer | Colorless clear liquid | 10 mL | 10×10 mL | -20°C | 100 µL |
A1508155C | D-luciferin | Yellow liquid | 0.1 mL | 10×0.1 mL | -20°C, Protect from light | On request |
A1508155D | Luciferase | Colorless clear liquid | 0.2 mL | 10×0.2 mL | -20°C | On request |
A1508155E | Cofactor | Colorless clear liquid | 0.1 mL | 10×0.1 mL | -20°C | On request |
A1508155F | ATP Standard (500µM) | Colorless clear liquid | 0.1 mL | 10×0.1 mL | -20°C | On request |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0838310 |
分析证书 | A1508155 |
| ZJ26F0535641 |
分析证书 | A1508155 |
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