本产品是一种基于XTT[2,3-双(2-甲氧基-4-硝基-5-磺苯基)-2H-四唑-5-甲酰胺内盐] 的广泛应用于细胞增殖和细胞毒性的快速、高灵敏度检测试剂盒。XTT在电子耦合试剂存在的情况下，被活细胞内产生的还原性脱氢酶还原成水溶性的橙黄色甲臜产物，可使用酶标仪在450 nm波长处进行吸光度检测，细胞活力越强甲臜生成的越多，颜色越深，一定范围内，吸光度与细胞活力成正比，因此可通过XTT进行细胞增殖和毒性分析。
本产品操作简单，仅需将XTT Labeling Reagent与Electron-coupling Reagent配制成的工作溶液直接加入到细胞中，37℃孵育一定时间，无需收集细胞，也无需洗涤细胞，直接使用酶标仪在450 nm波长处测定吸光度即可。
本产品可广泛用于细胞增殖测定（例如对生长因子、细胞因子等细胞诱导增殖研究）；细胞毒性测定（例如对抗癌药物产生的细胞毒性研究）等。
实验前准备
1.试剂&耗材
PBS、可用于吸光度检测的96孔细胞培养板（或其他细胞培养板）、细胞。
2.仪器
酶标仪(含450 nm滤光片)、CO2培养箱等。
使用方法
1.XTT工作液配制：
将XTT Labeling Reagent和Electron-coupling Reagent按照50:1的比列混合，配制成XTT工作液，配制好的溶液放置在冰上备用（现配现用）。
2.标准曲线制作（可选）：
按照一定的梯度将细胞接种于96孔细胞培养板中（如每孔100 μL培养液中分别含0，2000，4000，8000，16000，32000，64000个细胞），一般设置5~8个细胞数量梯度，每组做3~6个重复。
3.细胞培养
a 取生长状态良好的细胞，调整细胞密度，按每孔100 μL细胞悬液接种于96孔板中，同时设空白孔（不加细胞但是加入同体积的培养基）；
b 放置在细胞培养箱（37℃，5% CO2）中培养24-48小时；
c 根据实验设计对细胞进行培养和处理。
4. 细胞活力检测：
a 每孔中加入50 μL的XTT工作液，混匀；
b 放置在细胞培养箱（37℃，5% CO2）中培养2-4小时；
注：孵育时间随着独立的实验设置而变化（如所使用的细胞类型及细胞浓度）。因此，我们推荐按描述的方法在加入XTT工作液之后每隔1小时用酶标仪检测吸光度，选取吸光度数值较为适宜的时间点用于后续的实验。
5. 检测：测定450 nm各孔的吸光值，如无450 nm滤光片，可以使用450-490 nm的滤光片。
6. 细胞活力计算：
细胞活力（%）=（A-C）/（B-C）×100
抑制率（%）=（B-A）/（B-C）×100
A为实验组吸光度：实验组含有特定药物处理的细胞、培养基、XTT检测工作液。
B为对照组吸光度：对照组含有不加药物处理的细胞、培养基、XTT检测工作液。
C为空白组吸光度：空白组不含细胞，含有培养基、XTT 检测工作液。
注意事项
1. XTT Reagent 与 Electron Coupling Reagent 需避光保存，实验过程中注意避光操作。
2. 正式实验前请选取几个样本做预实验，以优化实验条件，确定最佳的细胞用量，取得最佳实验效果。
3. 试剂避免反复冻融，冻存后溶解时注意重新混匀。
4. 由于使用 96 孔板进行检测，如果细胞培养时间较长，一定要注意蒸发的问题。可以采取弃用周围一圈的办法，改加PBS，水或培养液；也可以把96孔板置于靠近培养箱内水源的地方，以缓解蒸发。
5. 接种时注意细胞悬液一定要混匀，以避免细胞沉淀下来，导致每孔中的细胞数量不等，可以每接种几个孔就混匀一下。
6. 用酶标仪检测前确保孔内没有气泡，否则会干扰测定。
7. 样品或试剂被细菌或真菌污染或试剂交叉污染可能会导致错误的结果。
8. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
This product is a rapid and highly sensitive detection kit based on XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazole-5-formamide inner salt], which is widely used for cell proliferation and cytotoxicity analysis. When XTT is present in an electron-coupling reagent, it is reduced by the reducing dehydrogenase produced within living cells to a water-soluble orange-yellow metachrome product. This can be detected by an enzyme reader at a wavelength of 450 nm. The stronger the cell viability, the more metachrome is generated, and the darker the color. Within a certain range, the absorbance is proportional to cell viability. Therefore, cell proliferation and cytotoxicity analysis can be conducted using XTT.
This product is simple to operate. Just mix the XTT Labeling Reagent and the Electron-coupling Reagent into a working solution and add it directly to the cells. Incubate at 37°C for a certain period of time. There is no need to collect the cells or wash them. Just use the enzyme reader to measure the absorbance at a wavelength of 450 nm directly.
This product can be widely used for cell proliferation determination (such as studies on cell induction proliferation by growth factors, cytokines, etc.) and cytotoxicity determination (such as studies on the cytotoxicity of anti-cancer drugs).
Note: The 500T package of this kit can test 500 samples; the 1000T package can test 1000 samples.
Preparation before the experiment
1.Reagents & Consumables
PBS、96-well cell culture plates (or other cell culture plates) for absorbance measurement, cells.
2.Instruments
Microplate reader (with 450 nm filter), CO2 incubator, etc.
Usage method
1.Preparation of XTT working solution:
Mix XTT Labeling Reagent and Electron-coupling Reagent in a ratio of 50:1 to prepare the XTT working solution. Place the prepared solution on ice for later use (use immediately after preparation).
2.Standard Curve Preparation (Optional):
The cells were inoculated into 96-well cell culture plates at certain gradients (for example, each well contained 100 μL of culture medium with 0, 2000, 4000, 8000, 16000, 32000, and 64000 cells respectively). Generally, 5 to 8 cell quantity gradients were set up, and 3 to 6 replicates were conducted for each group.
3.Cell culture:
a Select cells in a healthy growth state, adjust the cell density, and inoculate 100 μL of cell suspension per well into a 96-well plate. At the same time, set a blank well (without adding cells but adding the same volume of culture medium);
b Place it in the cell culture box (at 37℃, 5% CO2) for 24-48 hours of cultivation;
c According to the experimental design, cultivate and treat the cells.
4. Cell Viability Assay:
a Add 50 μL of the XTT working solution to each well and mix well；
b Place in the cell culture box (37°C, 5% CO2) and incubate for 2-4 hours;
Note: The incubation time varies depending on the independent experimental setup (such as the type of cells used and the cell concentration). Therefore, we recommend following the described method to measure the absorbance using an enzyme reader every 1 hour after adding the XTT working solution, and select the appropriate time point with suitable absorbance values for the subsequent experiments.
5. Detection:
Measure the absorbance values of each well at 450 nm. If there is no 450 nm filter, a 450-490 nm filter can be used.
6. Cell vitality calculation:
Cell viability (%) = (A - C) / (B - C) × 100
Inhibition rate (%) = (B - A) / (B - C) × 100
A represents the absorbance of the experimental group: the experimental group contains cells treated with a specific drug, culture medium, and XTT detection solution.
B represents the absorbance of the control group: the control group contains cells without drug treatment, culture medium, and XTT detection solution.
C represents the absorbance of the blank group: the blank group contains no cells, but contains culture medium and XTT detection solution.
Matters needing attention
1. The XTT Reagent and Electron Coupling Reagent should be stored in a dark place. During the experiment, be sure to operate in a dark environment.
2. Before conducting the formal experiment, select several samples for a preliminary test to optimize the experimental conditions and determine the optimal cell dosage for achieving the best experimental results.
3. The reagents should not be repeatedly frozen and thawed. When thawing for dissolution, make sure to remix thoroughly.
4. Since the detection is carried out using a 96-well plate, if the cell culture time is long, be sure to pay attention to the issue of evaporation. You can adopt the method of discarding the surrounding area and replacing it with PBS; or place the 96-well plate near the water source in the incubator to alleviate evaporation. When inoculating, make sure to mix the cell suspension thoroughly to avoid cell precipitation, which may result in unequal cell counts in each well. You can mix the suspension after inoculating several wells.
5. When inoculating, pay attention to thoroughly mixing the cell suspension to avoid cell precipitation, which may cause unequal cell counts in each well. Mix the suspension after inoculating several wells.
6. Before using the spectrophotometer for detection, ensure that there are no bubbles in the wells. Otherwise, it will interfere with the measurement.
7. Contamination of samples or reagents by bacteria or fungi or cross-contamination of reagents may lead to incorrect results.
8. For your safety and health, please wear a lab coat and wear disposable gloves.
ZJ26F0333434
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