BioReagent,即用型,用于化学发光 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。
ATP是细胞能量代谢的核心分子，其含量直接反映生物体及各器官的能量状态。作为关键的能源物质，ATP参与并调控着细胞的各种生理与病理活动，其水平波动会显著影响细胞功能。通常情况下，细胞凋亡、坏死或处于毒性状态时，ATP水平会降低；而在高葡萄糖等特定刺激下，某些细胞的ATP水平则可能升高。ATP的下降往往提示线粒体功能受损，在细胞凋亡过程中常伴随线粒体膜电位的降低。
本试剂盒基于萤火虫荧光素酶的催化原理进行设计：该酶在催化荧光素发光时需消耗ATP作为能源。当反应体系中酶与底物均过量时，荧光强度与ATP浓度在特定范围内呈正比关系，从而实现对溶液、细胞或组织样本中ATP含量的高灵敏度定量检测。
本试剂盒还可通过测定活细胞样品释放出的ATP含量，进而估算细胞浓度。此方法选择性计数活体细胞的数量，因为细胞死亡时，其 ATP 会迅速降解。通常，每个活细胞含有约 1 pg (10⁻¹² g) 或约 2 fmol (2 × 10⁻¹⁵ mol) ATP。对于特定的细胞系和生长培养基，可通过查阅文献，或在测定 ATP 含量前通过细胞染色并在显微镜下计数活细胞，获得更精确的每细胞 ATP 比值。使用所述方法，本试剂盒可测量多至 5 × 10⁵ 个活体细胞（样品密度为2 × 107 cell/ml）所释放的 ATP。其灵敏度远优于使用血球计数板显微镜检测法（后者通常仅能检测到约 2 × 10⁵ cell/ml）。
R1375244 | 组件 | 100T | 200 T | 储存温度 | Quantity Per Test |
R1375244A | ATP检测裂解液 | 50 mL | 100 mL | -20℃ | 200μL |
R1375244B | ATP标准溶液, 0.5mM | 0.1 mL | 0.2 mL | -20℃，避光 | 20μL |
R1375244C | ATP检测溶液 | 10 mL | 20 mL | -20℃，避光 | 100μL |
注意事项
1. 本试剂盒的检测试剂中含有荧光素酶，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有ATP污染。
2. 荧光素酶的活性对温度比较敏感，所以反应前细胞和ATP检测试剂均需平衡至室温后再进行测定。请勿室温存放。
3. ATP，特别是裂解后样品中的ATP在室温不太稳定，需在4℃或冰上操作。
4. 检测时需使用适合于细胞培养的白色或黑色的96孔板或384孔板。如果使用普通透明的96孔板或384孔板，相邻孔之间会产生相互干扰。
5. 请使用 0.2 µm 过滤的超纯水（电阻率 17 MΩ·cm 或等效规格）。
6. 如果需要稀释样品溶液或溶解固体样品，建议使用0.2µm过滤的超纯水或pH值约为7.8的稀缓冲液。建议不要使用砷酸盐作为样品缓冲液，因为它倾向于通过淬灭降低灵敏度。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。ATP 的 Kᴍ 值随离子强度的增加而增加。
7. 相对于简单的 ATP 含量检测，添加细胞培养基不会对本试剂盒检测试剂产生影响。若培养基额外添加组分过于复杂，可进行预实验检测。
8. 本试剂盒提供的ATP检测裂解液可以有效裂解并释放常见的培养细胞和组织中的ATP。对于一些特殊的组织或样品，如果发现检测出来的ATP水平显著低于预期水平，可以在裂解样品后并且在离心前，取部分样品煮沸2分钟以充分释放ATP。煮沸后样品中的蛋白质会变性，从而会在后续的离心步骤中被沉淀，因此煮沸的样品不能用于蛋白浓度测定、SDS-PAGE和Western检测。可以使用剩余的部分样品进行蛋白浓度测定、SDS-PAGE和Western检测。
9. 最终的实验结果与试剂的有效性、实验者的相关操作以及实验环境等因素密切相关，对此一定要重视！
使用方法一（标曲法）
1. 样品测定的准备：(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微升裂解液的比例(即相当于细胞培养液量2毫升的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 12000g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微升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旋涡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 12000g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毫克组织加入约100-200微升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 12000g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10µM这几个浓度，在后续的实验中，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。
3. ATP浓度的测定：
(1) 加100微升ATP检测试剂到检测孔内，室温放置3-5min。
(2) 在检测孔内加上20微升样品或已稀释好的ATP标准溶液。
(3) 用化学发光仪测定RLU值。
注：样品的体积可以自行在10-100微升范围内调节。如果样品中的ATP浓度比较低则可以加入100 微升样品，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裂解液稀释样品后再测定。
使用方法二（内标法）
1. 对于悬浮细胞
(1) 样品处理：
对于悬浮细胞，可直接取样进行后续测定
(2) ATP含量测定：
①向反应孔中加入100μL ATP检测溶液，静置10min或恢复至室温；
②测定孔：取50μL悬浮样品于EP管中，加入50μL纯水和100μL检测裂解液，充分混匀，裂解5-10min。裂解后取100μL加入到反应孔中混匀；
③内标孔：取50μL悬浮样品于EP管中，加入50μL ATP标准溶液作为内标和100μL检测裂解液，充分混匀，裂解5-10min。裂解后取100μL加入到反应孔中混匀；
【注意】为获得最佳结果，所加标准品中的 ATP 量应与细胞样品中测得的 ATP 量大致相同。即样品加内标发出的光应大约是样品单独发出的光的两倍。
④将板孔在室温继续孵育 5-25min，使发光信号趋于稳定。也可以每隔 5min 左右测定一次，取信号趋于平稳的时间点的数值作为实验数据。
⑤使用具有检测化学发光功能的多功能酶标仪进行化学发光检测。请根据仪器要求设置相 应的参数，记录发光值。
(3) ATP含量计算
细胞样品中的 ATP 量可通过以下公式计算：
其中：
ATP(SAM) 是细胞样品中的 ATP（摩尔数）
ATP(IS) 是所加内标中的 ATP（摩尔数）
L(SAM) 是细胞样品发出的光即测定孔的发光值
L(SAM+IS) 是细胞样品加内标发出的光即内标孔的发光值
(4) 活细胞浓度计算(若无此需求，可忽略)
如果已知每细胞的 ATP 量（通过实验或从文献中获得），则原始样品中每毫升的活细胞数可通过以下公式估算：
2. 对于贴壁细胞
(1) 样品处理：
吸除培养液，按照6孔板每孔加入200μL裂解液的比例(即相当于细胞培养液量2mL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，裂解5-10min，用于后续的测定。
(2) ATP含量测定：
①向反应孔中加入100μL ATP检测溶液，静置10min或恢复至室温；
②测定孔：取50μL裂解后样品于EP管中，加入150μL超纯水，充分混匀。取100μL加入到反应孔中混匀；
③内标孔：取50μL裂解后样品于EP管中，加入50μL ATP标准溶液作为内标和100μL超纯水，充分混匀。取100μL加入到反应孔中混匀；
④将板孔在室温继续孵育 5-25min，使发光信号趋于稳定。也可以每隔 5min 左右测定一次，取信号趋于平稳的时间点的数值作为实验数据。
⑤使用具有检测化学发光功能的多功能酶标仪进行化学发光检测。请根据仪器要求设置相 应的参数，记录发光值。
(3) ATP含量及活细胞浓度计算公式同上。
ATP is the core molecule of cellular energy metabolism, and its concentration directly reflects the energy status of an organism and its organs. As a crucial energy substance, ATP participates in and regulates various physiological and pathological processes within cells; fluctuations in its levels significantly impact cellular function. Typically, ATP levels decrease during cell apoptosis, necrosis, or under toxic conditions, while specific stimuli like high glucose can upregulate ATP levels in certain cells. A decline in ATP often indicates impaired mitochondrial function and, during apoptosis, frequently occurs concurrently with a decrease in mitochondrial membrane potential.
This kit is designed based on the catalytic principle of firefly luciferase: this enzyme requires ATP as an energy source to catalyze the light emission from luciferin. When both the luciferase and luciferin are in excess, the luminescence intensity is directly proportional to the ATP concentration within a certain range, enabling highly sensitive quantitative detection of ATP content in solutions, cells, or tissue samples.
This kit can also be used to estimate cell concentration by measuring the amount of ATP released from living cell samples. This method specifically counts viable cells, as ATP is rapidly degraded upon cell death. Typically, each viable cell contains approximately 1 pg (10⁻¹² g) or about 2 fmol (2 × 10⁻¹⁵ mol) of ATP. For a specific cell line and growth medium, a more accurate ATP-to-cell ratio can be obtained by consulting the literature or by staining cells and counting viable cells under a microscope before measuring ATP content. Using the described method, this kit can measure ATP released from up to 5 × 10⁵ viable cells (sample density of 2 × 10⁷ cells/mL). Its sensitivity is significantly superior to that of microscopy-based counting using a hemocytometer, which typically detects only about 2 × 10⁵ cells/mL.
R1375244 | Components | 100T | 200 T | Storage | Quantity Per Test |
R1375244A | ATP Assay Lysis Buffer | 50 mL | 100 mL | -20℃ | 200μL |
R1375244B | ATP Standard Solution, 0.5mM | 0.1 mL | 0.2 mL | -20℃, Store in the dark. | 20μL |
R1375244C | ATP Assay Reagent | 10 mL | 20 mL | -20℃, Store in the dark. | 100μL |
Precautions
1. The detection reagent in this kit contains luciferase. Repeated freeze-thaw cycles will lead to gradual loss of its activity. For optimal results, after the first thaw, it can be appropriately aliquoted for storage. However, ensure the aliquoting containers are free from ATP contamination.
2. Luciferase activity is sensitive to temperature. Therefore, both cells and the ATP detection reagent must be equilibrated to room temperature before the assay. Do not store the reagent at room temperature long-term.
3. ATP, especially in lysed samples, is relatively unstable at room temperature. Operations should be performed on ice or at 4°C.
4. Use white- or black-walled 96-well or 384-well plates suitable for cell culture for detection. Using standard clear plates may cause signal crosstalk between adjacent wells.
5. Use 0.2 µm filtered ultrapure water (resistivity 17 MΩ·cm or equivalent specification).
6. If dilution of sample solutions or dissolution of solid samples is required, the use of 0.2 µm filtered ultrapure water or a dilute buffer with a pH of approximately 7.8 is recommended. The use of arsenate as a sample buffer is not recommended, as it tends to reduce sensitivity through quenching. Additionally, high salt concentrations in samples can inhibit luciferase and decrease sensitivity. The Kₘ value for ATP increases with ionic strength.
7. The addition of cell culture medium does not affect the detection reagent of this kit compared to simple ATP detection. If the medium contains unusually complex additional components, a preliminary test may be performed.
8. The ATP Assay Lysis Buffer provided effectively lyses common cultured cells and tissues to release ATP. For certain specific tissues or samples, if the detected ATP level is significantly lower than expected, a portion of the lysate can be boiled for 2 minutes after lysis but before centrifugation to fully release ATP. Boiling will denature proteins, which will precipitate during subsequent centrifugation. Therefore, boiled samples cannot be used for protein concentration determination, SDS-PAGE, or Western blotting. Use the remaining, unboiled portion of the sample for these analyses.
9. The final experimental results are closely related to factors such as reagent validity, the operator's technique, and the experimental environment. It is essential to pay close attention to these factors.
Instructions for Use 1 (Standard Curve Method)
1. Sample Preparation (Note: Perform cell/tissue lysis on ice or at 4°C):
(1) For Adherent Cells:
Aspirate the culture medium. Add lysis buffer proportional to 200 μL per well of a 6-well plate (i.e., approximately 1/10 of the 2 mL culture medium volume). Lyse the cells by pipetting up and down repeatedly or gently rocking the plate to ensure complete contact. Cells typically lyse immediately upon contact with the buffer. Centrifuge the lysate at 12,000× g for 5 minutes at 4°C. Collect the supernatant for subsequent assay.
(2) For Suspension Cells:
Centrifuge to pellet the cells, discard the supernatant, and gently resuspend the pellet. Add lysis buffer proportional to 200 μL per well of a 6-well plate (based on cell count). Lyse the cells by flicking the tube or brief vortexing to ensure complete contact. Cells typically lyse immediately. Centrifuge the lysate at 12,000× g for 5 minutes at 4°C. Collect the supernatant for subsequent assay.
(3) For Tissue Samples:
Add approximately 100-200 μL of lysis buffer per 20 mg of tissue. Homogenize using a glass homogenizer or other appropriate device. Thorough homogenization ensures complete lysis. Centrifuge the homogenate at 12,000× g for 5 minutes at 4°C. Collect the supernatant for subsequent assay.
2. Standard Curve Preparation:
Thaw the necessary reagents on ice. Dilute the ATP Standard Solution with the ATP Assay Lysis Buffer to create an appropriate concentration gradient. The specific concentrations depend on the expected ATP levels in your samples. For initial experiments, concentrations like 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 µM can be tested. Adjust the range in subsequent experiments based on sample ATP concentrations.
3. ATP Concentration Measurement:
(1) Add 100 μL of the ATP Detection Reagent to each well of the assay plate. Incubate at room temperature for 3-5 minutes.
(2) Add 20 μL of the sample or the diluted ATP standard solution to the wells containing the reagent.
(3) Measure the Relative Light Units (RLU) using a luminometer.
Note: The sample volume can be adjusted within the range of 10-100 μL. If the ATP concentration in the sample is low, add up to 100 μL. If the concentration is high, use a smaller volume, ensuring the same volume is used for the standard curve dilutions. If the ATP concentration is exceptionally high, dilute the sample with ATP Assay Lysis Buffer before measurement.
Instructions for Use 2 (Internal Standard Method)
1. For Suspension Cells
(1) Sample Processing:
For suspension cells, samples can be directly taken for subsequent measurement.
(2) ATP Measurement:
① Add 100 μL of ATP detection solution to the required reaction wells. Let stand for 10 min or until equilibrated to room temperature.
② Sample Wells: Transfer 50 μL of the suspended cell sample into a microcentrifuge tube. Add 50 μL of pure water and 100 μL of detection lysis buffer. Mix thoroughly and lyse for 5-10 min. After lysis, transfer 100 μL of the mixture to a reaction well and mix.
③ Internal Standard Wells: Transfer 50 μL of the suspended cell sample into a microcentrifuge tube. Add 50 μL of ATP standard solution (as an internal standard) and 100 μL of detection lysis buffer. Mix thoroughly and lyse for 5-10 min. After lysis, transfer 100 μL of the mixture to a reaction well and mix.
Note: For optimal results, the amount of ATP spiked as the internal standard should be approximately equal to the amount of ATP present in the cell sample. The luminescence from the sample plus internal standard should be roughly double that of the sample alone.
④ Continue to incubate the plate at room temperature for 5-25 min to allow the luminescent signal to stabilize. Alternatively, measurements can be taken approximately every 5 min, using the value at the time point when the signal stabilizes as the experimental data.
⑤ Measure chemiluminescence using a multimode microplate reader equipped with chemiluminescence detection capability. Set the appropriate parameters according to the instrument's instructions and record the luminescence values.
(3) ATP Calculation
The amount of ATP in the cell sample can be calculated using the following formula:
Where:
ATP(SAM) is the amount of ATP in the cell sample (in moles)
ATP(IS) is the amount of ATP spiked as the internal standard (in moles)
L(SAM) is the luminescence emitted by the cell sample (value from the Sample Well)
L(SAM+IS) is the luminescence emitted by the cell sample plus the internal standard (value from the Internal Standard Well)
(4) Viable Cell Concentration Calculation (Omit if not required)
If the ATP content per cell is known (determined experimentally or obtained from literature), the number of viable cells per milliliter in the original sample can be estimated using the following formula:
2. For Adherent Cells
(1) Sample Processing:
Aspirate and discard the culture medium. Add lysis buffer to the wells at a ratio of 200 μL per well for a 6-well plate (equivalent to 1/10 of the 2 mL culture volume). To ensure complete lysis, use a pipette to repeatedly triturate or rock the plate to allow the lysis buffer to fully contact and lyse the cells. Lyse for 5-10 min before proceeding with the measurement.
(2) ATP Measurement:
① Add 100 μL of ATP detection solution to the required reaction wells. Let stand for 10 min or until equilibrated to room temperature.
② Sample Wells: Transfer 50 μL of the lysed sample into a microcentrifuge tube. Add 150 μL of ultrapure water and mix thoroughly. Transfer 100 μL of this mixture to a reaction well and mix.
③ Internal Standard Wells: Transfer 50 μL of the lysed sample into a microcentrifuge tube. Add 50 μL of ATP standard solution (as an internal standard) and 100 μL of ultrapure water. Mix thoroughly. Transfer 100 μL of this mixture to a reaction well and mix.
④ Continue to incubate the plate at room temperature for 5-25 min to allow the luminescent signal to stabilize. Alternatively, measurements can be taken approximately every 5 min, using the value at the time point when the signal stabilizes as the experimental data.
⑤ Measure chemiluminescence using a multimode microplate reader equipped with chemiluminescence detection capability. Set the appropriate parameters according to the instrument's instructions and record the luminescence values.
(3) ATP Calculation and Viable Cell Concentration Calculation:
Use the same formulas provided above in sections 1(3) and 1(4).
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