胆固醇(Cholesterol)，又称胆甾醇，不仅是哺乳动物细胞膜的重要组成部分，同时也是合成胆汁酸，维生素D以及甾体激素的重要原料。胆固醇广泛参与胞内转运、细胞信号转导等细胞功能，并维持着膜结构的流动性。在正常情况下，机体在肝脏中合成或从食物中摄取的胆固醇，转化为甾体激素或成为细胞膜的组分，并使血液中胆固醇的浓度保持相对稳定。由于血液总胆固醇含量的升高与动脉粥样硬化、心血管疾病存在一定的关联性。所以对血液中胆固醇含量进行检测具有重要的临床意义。
本试剂盒的检测原理为。胆固醇酯首先在胆固醇酯酶的催化作用下水解生成游离胆固醇和脂肪酸，游离胆固醇进一步在胆固醇氧化酶的作用下和氧气发生氧化反应生成H2O2和胆甾烯酮。在辣根过氧化物酶存在的情况下，Amplex Red能与H2O2反应，产生强烈的红色荧光物质试卤灵。试卤灵的最大激发波长为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荧光和吸光度两种方法来进行检测。如果检测时不使用胆固醇酯酶，那么检测出来的就是游离胆固醇的含量。测定出总胆固醇和游离胆固醇的含量，二者相减就可以计算出胆固醇酯的含量。
采用吸光度检测时，在样品体积为50 μL的情况下可检测低至3.125 µM的胆固醇，在3.125-400 µM浓度范围内有良好的线性关系。采用荧光检测时，在样品体积为50 μL的情况下可检测低至0.23 µM的胆固醇，在0.23-30 µM浓度范围内有良好的线性关系。
使用方法
1. 样本制备
（1）血液样品的准备：对于血清样品，将全血在常温下静置30分钟-2小时，4ºC约1000-2000×g离心10分钟，取上清；对于血浆样品，将全血用肝素或者EDTA进行抗凝，4ºC约1000-2000×g离心10分钟，取上清。
（2）细胞样本的准备：对于培养的贴壁细胞，PBS洗涤细胞一次。对于培养的悬浮细胞，离心收集细胞用PBS洗涤细胞一次。按照每100万细胞加入200 μL 裂解液的比例加入裂解液，适当吹打，冰浴10分钟充分裂解细胞。4ºC, 12,000×g离心5分钟，取上清用于后续检测。
（3）对于组织样品，按照每10 mg组织加入100 μL 裂解液的比例，使用匀浆器冰浴匀浆。4ºC约12,000×g离心5分钟，取上清用于后续检测。
（4）细胞培养上清样品的准备：对于贴壁细胞，直接取培养液；对于悬浮细胞，离心取培养液。
以上所有操作均需在4ºC或冰上操作。制备好的细胞或组织样品如果不能立即检测，可以-20ºC或-80ºC冻存。
2. 标准曲线的设置
（1）吸光度检测：取9支EP管，第一支EP管中加入184 μL裂解液或检测缓冲液(如果检测的是用裂解液制备的细胞或组织裂解样品，使用裂解液；如果检测的是血液、上清等无需裂解处理的样品，使用检测缓冲液)，其他每管中加入 100 μL。取16 μL 胆固醇标准溶液 (5 mM)到第一支EP管中，混匀后吸取100 μL第2支EP管，按此步骤往后依次稀释，最后一支EP管为100 μL 裂解液或检测缓冲液。此时，标准品的浓度依次为400、200、100、50、25、12.5、6.25、3.125、0 µM。吸光度检测时建议使用透明96孔板。
（2）荧光检测：取9支EP管，第一支EP管中加入497 μL裂解液或检测缓冲液(如果检测的是用裂解液制备的细胞或组织裂解样品，使用裂解液；如果检测的是血液、上清等无需裂解处理的样品，使用检测缓冲液)，其他每管中加入 100 μL。取3 μL 胆固醇标准溶液 (5 mM)到第一支EP管中，混匀后吸取100 μL第2支EP管，按此步骤往后依次稀释，最后一支EP管为100 μL 裂解液或检测缓冲液。此时，标准品的浓度依次为30、15、7.5、3.75、1.88、0.94、0.47、0.23、0 µM。荧光检测时建议使用96孔黑板。
3. 显色工作液的配制
按照每个检测反应50 μL的体积配制显色工作液。根据待检测样品(包括标准品)的数量，配制适量的显色工作液。按照(1)和(2)表配置反应液，分别检测总胆固醇和游离胆固醇的含量，从总胆固醇的含量中减去游离胆固醇的含量即可获得胆固醇酯的含量。
在制作标准曲线时，加入标准品孔中的检测工作液须加胆固醇酯酶，即按照总胆固醇的检测的工作液进行配制。此外，H2O2的存在会对胆固醇的检测产生干扰。如果样品含有H2O2，须同时设置样品背景对照孔，加入不含酶混合物的显色工作液，即配制显色工作液时酶混合物用检测缓冲液替代。计算时样品孔的读数需要减去样品背景对照孔的读数。
（1）总胆固醇的检测
样本数 | 1 | 10 | 20 | 50 | 检测缓冲液 | 32 μL | 320 μL | 640 μL | 1600 μL | Amplex Red | 8 μL | 80 μL | 160 μL | 400 μL | 胆固醇酯酶 | 2 μL | 20 μL | 40 μL | 100 μL | 酶混合物 | 8 μL | 80 μL | 160 μL | 400 μL | 总体积 | 50 μL | 500 μL | 1000 μL | 2500 μL |
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（2）游离胆固醇的检测
样本数 | 1 | 10 | 20 | 50 | 检测缓冲液 | 34 μL | 340 μL | 680 μL | 1700 μL | Amplex Red | 8 μL | 80 μL | 160 μL | 400 μL | 酶混合物 | 8 μL | 80 μL | 160 μL | 400 μL | 总体积 | 50 μL | 500 μL | 1000 μL | 2500 μL |
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4. 样本测定
（1）取1-50 μL样品或稀释后的样品至96孔板中，并相应地加入裂解液或检测液补足至50 µL。同时设置仅含裂解液或检测缓冲液的孔为空白对照孔。
（2）各孔加入检测工作液50 μL，混匀。37ºC避光反应30分钟。如果由于样品中胆固醇或胆固醇酯含量偏低而导致吸光度偏低或荧光偏弱，可适当延长反应时间；对于胆固醇酯的检测，如果胆固醇酯的含量偏高，建议延长反应时间至60分钟或更长时间以确保胆固醇酯的充分水解，或者适当稀释样品。
（3）如果使用吸光度检测，测定A570；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
建立标准曲线，并计算样品中总胆固醇/游离胆固醇的浓度(A)，如果样品的背景对照孔信号比较高，样品的信号值应减去样品背景对照孔的信号值。总胆固醇/游离胆固醇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胆固醇浓度(μM)；n为样品总稀释倍数。总胆固醇的含量减去游离胆固醇的含量即可获得胆固醇酯的含量；也可以根据胆固醇的分子量386.7计算出质量浓度(μg/mL) = C × 0.3867。
注意事项
1. Amplex Red在空气中不太稳定，开启后应尽快使用，且在使用过程中一定要注意适当避光。
2. Amplex Red的反应产物在还原剂的存在下会很不稳定，因此最终反应体系中的二硫苏糖醇(DTT)、β-巯基乙醇或类似还原剂的浓度应低于10 μM。
3. 请确保反应体系的pH值在7-8之间，否则会影响Amplex Red的稳定性和荧光值。
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96孔黑板。
Cholesterol, also known as cholesterin, is not only an important component of mammalian cell membranes but also a crucial precursor for the synthesis of bile acids, vitamin D, and steroid hormones. Cholesterol is widely involved in cellular functions such as intracellular transport and signal transduction, and maintains the fluidity of membrane structures. Under normal conditions, cholesterol synthesized in the liver or obtained from the diet is converted into steroid hormones or incorporated into cell membranes, keeping the concentration of cholesterol in the blood relatively stable. Since elevated total cholesterol levels in the blood are associated with atherosclerosis and cardiovascular diseases, the detection of cholesterol levels in the blood is of significant clinical importance.
The detection principle of this kit is as follows: Cholesterol esters are first hydrolyzed by cholesterol esterase to produce free cholesterol and fatty acids. Free cholesterol is then oxidized by cholesterol oxidase in the presence of oxygen to generate H₂O₂ and cholestenone. In the presence of horseradish peroxidase, Amplex Red reacts with H₂O₂ to produce the highly fluorescent red product resorufin. Resorufin 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 detection by both fluorescence and absorbance methods. If cholesterol esterase is omitted during detection, the assay measures free cholesterol content. By determining total cholesterol and free cholesterol levels, the cholesterol ester content can be calculated by subtraction.
When using absorbance detection, with a sample volume of 50 μL, this kit can detect cholesterol concentrations as low as 3.125 µM, and exhibits a strong linear relationship within the concentration range of 3.125–400 µM. When using fluorescence detection, with a sample volume of 50 μL, this kit can detect cholesterol concentrations as low as 0.23 µM, and exhibits a strong linear relationship within the concentration range of 0.23–30 µM.
Usage Protocol
1. Sample Preparation
1) Preparation of Blood Samples:
For serum samples: Allow whole blood to clot at room temperature for 30 minutes to 2 hours. Centrifuge at approximately 1000-2000 × g for 10 minutes at 4°C and collect the supernatant.
For plasma samples: Collect whole blood using heparin or EDTA as an anticoagulant. Centrifuge at approximately 1000-2000 × g for 10 minutes at 4°C and collect the supernatant.
2) Preparation of Cell Samples:
For cultured adherent cells: Wash the cells once with PBS; For cultured suspension cells: Centrifuge to collect the cells and wash them once with PBS. Add Lysis Buffer at a ratio of 200 µL per 1 million cells. Pipette appropriately to mix and incubate on ice for 10 minutes for complete lysis. Centrifuge at 12,000 × g for 5 minutes at 4°C. Collect the supernatant for subsequent assays.
3) Preparation of Tissue Samples:
Add Lysis Buffer at a ratio of 100 µL per 10 mg of tissue. Homogenize the tissue using a homogenizer on ice. Centrifuge at approximately 12,000 × g for 5 minutes at 4°C. Collect the supernatant for subsequent assays.
4) Preparation of Cell Culture Supernatant Samples:
For adherent cells: Directly collect the culture medium; For suspension cells: Centrifuge the culture and collect the supernatant.
All the above procedures must be performed at 4°C or on ice. Prepared cell or tissue samples can be stored at -20°C or -80°C if not assayed immediately.
2. Establishment of Standard Curve
1) Absorbance Detection: Take nine EP tubes. Add 184 μL of lysis buffer or assay buffer (use lysis buffer for lysed cell or tissue samples prepared with lysis buffer; use assay buffer for samples such as blood or supernatant that do not require lysis) to the first EP tube, and add 100 μL to each of the remaining tubes. Pipette 16 μL of Cholesterol Standard Solution (5 mM) into the first EP tube, mix thoroughly, then transfer 100 μL to the second EP tube. Perform serial dilutions in the same manner to the subsequent tubes, with the last tube containing 100 μL of lysis buffer or assay buffer. The resulting standard concentrations are 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, and 0 µM, respectively. For absorbance detection, a transparent 96-well plate is recommended.
2) Fluorescence Detection: Take nine EP tubes. Add 497 μL of lysis buffer or assay buffer (use lysis buffer for lysed cell or tissue samples prepared with lysis buffer; use assay buffer for samples such as blood or supernatant that do not require lysis) to the first EP tube, and add 100 μL to each of the remaining tubes. Pipette 3 μL of Cholesterol Standard Solution (5 mM) into the first EP tube, mix thoroughly, then transfer 100 μL to the second EP tube. Perform serial dilutions in the same manner to the subsequent tubes, with the last tube containing 100 μL of lysis buffer or assay buffer. The resulting standard concentrations are 30, 15, 7.5, 3.75, 1.88, 0.94, 0.47, 0.23, and 0 µM, respectively. For fluorescence detection, a black 96-well plate is recommended.
3. Preparation of Chromogenic Solution
Prepare the Color Developer Working Solution at a volume of 50 μL per assay reaction. Prepare an appropriate amount of the working solution based on the number of samples to be tested (including standards). Prepare the reaction solutions according to tables (1) and (2) for the determination of total cholesterol and free cholesterol, respectively. Subtract the free cholesterol content from the total cholesterol content to obtain the cholesterol ester content.
When generating the standard curve, the detection working solution added to the standard wells must include cholesterol esterase, i.e., prepare it according to the working solution used for total cholesterol determination. In addition, the presence of H₂O₂ may interfere with cholesterol detection. If the sample contains H₂O₂, a sample background control well must be set up in parallel. Add the Color Developer Working Solution without the enzyme mixture, i.e., replace the enzyme mixture with assay buffer when preparing the working solution. When calculating results, subtract the absorbance reading of the sample background control well from that of the sample well.
1）Total Cholesterol Detection
Samples | 1 | 10 | 20 | 50 | Assay Buffer | 32 μL | 320 μL | 640 μL | 1600 μL | Amplex Red | 8 μL | 80 μL | 160 μL | 400 μL | Cholesterol Esterase | 2 μL | 20 μL | 40 μL | 100 μL | Enzyme Mix | 8 μL | 80 μL | 160 μL | 400 μL | Total volume | 50 μL | 500 μL | 1000 μL | 2500 μL |
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2）Free Cholesterol Detection
Samples | 1 | 10 | 20 | 50 | Assay Buffer | 34 μL | 340 μL | 680 μL | 1700 μL | Amplex Red | 8 μL | 80 μL | 160 μL | 400 μL | Enzyme Mix | 8 μL | 80 μL | 160 μL | 400 μL | Total volume | 50 μL | 500 μL | 1000 μL | 2500 μL |
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4. Sample Assay
1) Add 1–50 μL of sample or diluted sample to a 96‑well plate, and add the corresponding lysis buffer or assay buffer to bring the volume to 50 μL. Set up wells containing only lysis buffer or assay buffer as blank control wells.
2) Add 50 μL of Assay Working Solution to each well and mix. Incubate at 37 °C in the dark for 30 minutes. If the absorbance or fluorescence is low due to low cholesterol or cholesterol ester content in the sample, the reaction time may be appropriately extended; for cholesterol ester detection, if the cholesterol ester content is high, it is recommended to extend the reaction time to 60 minutes or longer to ensure complete hydrolysis of cholesterol esters, or to dilute the sample appropriately.
3) For absorbance detection, measure A₅₇₀; for fluorescence detection, measure fluorescence intensity with excitation at 560 nm and emission at 590 nm.
5. Calculation of Results
Establish a standard curve and calculate the concentration of total cholesterol/free cholesterol in the sample (A). If the signal from the sample background control well is relatively high, the sample signal value should be subtracted by the signal value of the sample background control well. The formula for calculating the concentration of total cholesterol/free cholesterol is as follows: C (μM) = A × n
Note: A is the cholesterol concentration (μM) determined from the standard curve; n is the total dilution factor of the sample.
The content of cholesterol ester can be obtained by subtracting the free cholesterol content from the total cholesterol content. The mass concentration (μg/mL) can also be calculated based on the molecular weight of cholesterol (386.7):
Mass concentration (μg/mL) = C × 0.3867.
Precautions
1. Amplex Red is unstable in air. It should be used as soon as possible after opening, and proper protection from light must be maintained during use.
2. The reaction product of Amplex Red is unstable in the presence of reducing agents. Therefore, the concentration of dithiothreitol (DTT), β-mercaptoethanol, or similar reducing agents in the final reaction system should be kept below 10 μM.
3. Ensure that the pH of the reaction system is between 7 and 8; otherwise, the stability of Amplex Red and the fluorescence signal may be affected.
4. For absorbance detection, a transparent 96‑well plate is recommended; for fluorescence detection, a black 96‑well plate is recommended.
ZJ26F0434699
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