A2615380计算溶液所需的质量、体积或浓度。
为什么选择此级别
，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
避光,-20°C储存。超低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 1 篇同行评审文献引用。
概述
Live & DeadTM动物细胞活力/毒性检测试剂盒（Calcein AM, EthD-I）是一种为动物细胞死活检测提供双荧光染色的试剂盒。试剂盒内的两种探针可分别测定细胞内酯酶活性和质膜完整性从而反映细胞活力。本试剂盒可用于荧光显微镜、流式细胞仪、酶标仪以及其他荧光检测系统。 本试剂盒可以应用于大多数的真核哺乳动物细胞，包括贴壁细胞核的某些组织，但不适用于真菌和酵母。该试剂盒与相同用处的台盼蓝相比，更快捷安全且灵敏度更高。
物质分类：液态
组分：
产品参数：
Calcein AM: Ex/Em = 494/517 nm；EthD-I: Ex/Em = 528/617 nm（结合DNA）
使用方法：
荧光显微镜检测
1. 准备工作液
准备 2 μM Calcein AM 和 4 μM EthD-I 的染色工液：取出 Calcein AM 和 EthD-I 原液，使其恢复室温。将 20 μL 2 mM EthD-I 和 5 μL 4 mM Calcein AM 与 10 mL PBS 或其他无血清缓冲液或培养基混合，涡旋混匀。上述工作液可直接用于细胞染色。
注： Calcein AM 的水溶液易水解，应当天用完。 Calcein AM 和 EthD-I 的浓度选择依据所用细胞类型不同而有所区别，推荐浓度范围为 0.1~10 μM。
2. 准备细胞并开展实验
（1）对于贴壁细胞，可用 1×PBS 清洗 2~3 次后进行染色。对于悬浮细胞， 250- 1000×g 室温离心 5 min，收集细胞染色。
（2）用 1×PBS 充分清洗细胞 2~3 次，以充分去除残留的酯酶活性。
（3）对于贴壁细胞， 加入足够量的 Calcein AM/EthD-I 染色工作液。对于悬浮细胞， 加入适量的染色工作液， 使细胞密度控制在 1- 5×105/mL。
（4）室温避光孵育 15 min ~ 20 min (如果工作液浓度较高或者孵育温度较高，应适当的减少孵育时间)。
（5）荧光显微镜下观察标记的细胞。
流式细胞仪检测
1. 取出试剂，恢复至室温。
2. 准备 2 μM Calcein AM 和 4 μM EthD-I 的染色工作液：取出 Calcein AM 和 EthD-I 原液，恢复至室温。 将 20 μL 2 mMEthD-I 和 5 μL 4 mM Calcein AM 与 10 mL PBS 或其他无血清缓冲液或培养基涡旋混匀。工作液可直接于细胞染色。
3. 用 1×PBS 充分清洗细胞 2~3 次。
4. 用 0.5 mL 染色工作液悬浮细胞，控制细胞密度为 1-5×105/mL。
注： 推荐准备两管额外的细胞样品， 每管只加入一种染料（Calcein AM 和 EthD-I），用于流式单染的补偿调节；另准备一管 仅含缓冲液（该缓冲液与配制 Calcein AM 及 EthD-I 检测工作液的缓冲液宜保持一致）的细胞样品用作流式检测时的阴性对照。
5. 室温避光孵育 15-20 min。
6. 在 1-2 h 内，通过流式细胞仪检测细胞活性。 Calcein AM 可以由 488 nm 激光激发，检测荧光发射光谱约在 530 nm 处，EthD-I 发射光谱约在 610 nm 处。
注： 细胞圈门时，注意排除细胞碎片， 使用单染管调节补偿， 双染管流式检测应获得两个相对独立的细胞群： 显示绿色荧光的活细胞群和红色荧光的死细胞群。
酶标仪检测
1. 在 96 孔黑色酶标板中培养适量的贴壁或悬浮细胞。
注：用 1%的皂苷或 0.1-0.5%洋地黄皂苷处理细胞 10 min，即可得到死细胞。
2. 准备 2 μM Calcein AM 和 4 μM EthD-I 的染色工作液：
取出 Calcein AM 和 EthD-I 原液，使其恢复室温。将 20 μL 2 mM EthD-I 和 5 μL 4 mM Calcein AM 10 mL PBS 或其他无血清缓冲液或培养基混合，涡旋混匀。
注：（1）10 mL 的染色液足够染色一个 96 孔板，可根据实验需要调整染色液的体积。Calcein AM 和 EthD-I 的浓度可在 0.1~10μM 之间摸索。
（2）Calcein AM 的水溶液易水解，应当天用完。 EthD-I 工作液可保存在-20℃,至少可保存一年。
3. 用 1×PBS 充分清洗细胞，以充分去除残留的酯酶活性。对于贴壁细胞，每孔加 100 μL PBS 清洗细胞。对于悬浮细胞，加 100 μL PBS 重悬细胞，离心去上清。重复上述操作。
4. 每孔中加入 100 μL PBS。
5. 每孔中加入 100 μL 染色工作液， 使每孔总体积为 200 μL ，Calcein AM 的终浓度为 1 μM，EthD-I 的终浓度为 2 μM。轻轻摇晃培养板，使液体均匀覆盖细胞。
6. 室温避光孵育 30~45 min。
注：不同的细胞最佳孵育时间有所不同， 以 30min 作为初始孵育时间， 后续可以根据实际染色效果对染色时间进行适当调整和优化，以得到更加理想的染色效果。
7. 酶标仪检测。当酶标仪设置为 fluorescein 时， 可以检测 Calcein AM；当酶标仪设置为 rhodamine 或 Texas Red 时，可以检测 EthD-I。根据光谱特性选择最佳的发射及激发波长。
注：通过对比样品组与对照组测量的 Relative fluorescence values (RFU)，可以得出死细胞与活细胞数量的变化。下面也提供了另一种数据分析的方法。
计算某个区域活细胞与死细胞的比例下面方法可以计算出死细胞与活细胞的比例。 需要的样品有死细胞对照组、活细胞对照组及要测的样品组。用 1%的皂苷或 0.1- 0.5%洋地黄皂苷处理细胞 10 min ， 即可得到死细胞。
1. 准备染色工作液及按上述步骤染色细胞。另外，分别准备 1 mL 2 μM Calcein AM 和 4 μM EthD-I 溶液，按照下列指示来染色对照组。 对于下面组别的细胞或无细胞组，需要保持细胞数量、检测工作液浓度、孵育时间和孵育温度的完全一致。
2. 样品组及对照组测量：
A. 样品组在 645 nm 的测量值，记为 Calcein AM 和 EthD-I=F(645)sam。
B. 样品组在 530 nm 的测量值，记为 Calcein AM 和 EthD-I=F(530)sam。
C. 死细胞 EthD-I 单染对照组在 645 nm 的测量值，记为 EthD-I=F(645)max
D. 死细胞 Calcein AM 单染对照组在 645 nm 的测量值，记为 Calcein AM=F(645)min
E. 活细胞 EthD-I 单染对照组在 530 nm 的测量值，记为 EthD-I=F(530)min。
F. 活细胞 Calcein AM 单染对照组在 530 nm 的测量值，记为 Calcein AM=F(530)max。
G. 没有细胞的空白对照孔(加染料或不加染料均可) ，530 nm 处的检测值记为 F(530)0。
H. 没有细胞的空白对照孔(加染料或不加染料均可) ，645 nm 处的检测值记为 F(645)0。
3. 根据测量数据计算死细胞与活细胞的比例：
% Live Cells = (B - E) ÷(F - E)
% Dead Cells = (A - D) ÷(C - D)
确定某个区域活细胞与死细胞的比例
通过制作 530 nm 和 645 nm 处的荧光光谱标准曲线，可以确定死细胞与活细胞的数量，每个染料的荧光强度分别与样品中死细胞或活细胞的数量成直线型关系。
注意事项：
1. 使用前请将产品瞬时离心至管底，再进行后续实验。
2. 酚红或血清对于本试剂盒的检测有一定的干扰。
3. 荧光染料均存在淬灭问题，实验操作时请尽量注意避光，以减缓荧光淬灭。
4. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
应用范围：
死、活细胞染色（动物）
Live & deadtm animal cell viability / toxicity detection kit (calcein am, ethd-i) is a kit that provides double fluorescent staining for the detection of animal cell death and survival. The two probes in the kit can respectively measure the activity of cellular lactonase and the integrity of plasma membrane to reflect cell viability. The kit can be used for fluorescence microscopy, flow cytometry, microplate reader and other fluorescence detection systems. This kit can be applied to most eukarYOtic mammalian cells, including some tissues with adherent nuclei, but it is not applicable to fungi and yeast. Compared with trypan blue, the kit is faster, safer and more sensitive.
Component：
Product parameters：
Calcein am: ex/em = 494 / 517 nm; Ethd-i: ex/em = 528 / 617 nm (bound DNA)
Usage:
Fluorescence microscopy detection
1. Prepare working fluid
Preparation 2 μ M Calcein AM and 4 μ M EthD-I staining solution: Remove the original solution of Calcein AM and EthD-I and restore them to room temperature. Add 20 μ L 2 mM EthD-I and 5 μ Mix 4 mM Calcein AM with 10 mL PBS or other serum-free buffer or culture medium, vortex well. The above working solution can be directly used for cell staining.
Note: The aqueous solution of Calcein AM is easily hydrolyzed and should be used up every day. The concentration selection of Calcein AM and EthD-I varies depending on the type of cell used, with a recommended concentration range of 0.1-10 μ M.
2. Prepare cells and conduct experiments
(1) For adherent cells, they can be washed 2-3 times with 1 × PBS before staining. For suspended cells, centrifuge at room temperature of 250-1000 × g for 5 minutes and collect cells for staining.
(2) Wash the cells thoroughly 2-3 times with 1 × PBS to remove residual esterase activity.
(3) For adherent cells, add sufficient amount of Calcein AM/EthD-I staining solution. For suspended cells, add an appropriate amount of staining solution to control the cell density between 1-5 × 105/mL.
(4) Incubate at room temperature in dark for 15-20 minutes (if the working solution concentration is high or the incubation temperature is high, the incubation time should be appropriately reduced).
(5) Observe the labeled cells under a fluorescence microscope.
Flow cytometry detection
1. Remove the reagent and restore it to room temperature.
2. Preparation 2 μ M Calcein AM and 4 μ M EthD-I staining solution: Take out the original solution of Calcein AM and EthD-I, and restore to room temperature. Add 20 μ L 2 mMEthD-I and 5 μ Vortex mix 4 mM Calcein AM with 10 mL PBS or other serum-free buffer or culture medium. The working fluid can directly stain cells.
3. Wash cells thoroughly 2-3 times with 1 × PBS.
4. Suspend cells with 0.5 mL of staining solution and control the cell density to 1-5 × 105/mL.
Note: It is recommended to prepare two additional cell samples, each containing only one dye (Calcein AM and EthD-I), for compensatory regulation of flow cytometry single staining; Prepare another cell sample containing only buffer solution (which should be consistent with the buffer used to prepare Calcein AM and EthD-I detection working solutions) as a negative control for flow cytometry analysis.
5. Incubate at room temperature in dark for 15-20 minutes.
6. Within 1-2 hours, cell activity was detected by flow cytometry. Calcein AM can be excited by a 488 nm laser, with fluorescence emission spectra detected at around 530 nm and EthD-I emission spectra at around 610 nm.
Note: When using the cell circle gate, attention should be paid to excluding cell debris and using a single staining tube to regulate compensation. Double staining tube flow cytometry should obtain two relatively independent cell populations: a live cell population displaying green fluorescence and a dead cell population displaying red fluorescence.
ELISA reader detection
1. Cultivate an appropriate amount of adherent or suspended cells in a 96 well black ELISA plate.
Note: Dead cells can be obtained by treating cells with 1% saponin or 0.1-0.5% digitalis saponin for 10 minutes.
2. Preparation 2 μ M Calcein AM and 4 μ M EthD-I staining solution:
Remove the original solutions of Calcein AM and EthD-I and restore them to room temperature. Add 20 μ L 2 mM EthD-I and 5 μ Mix 4 mM Calcein AM 10 mL PBS or other serum-free buffer or culture medium, vortex well.
Note: (1) 10 mL of staining solution is sufficient to stain a 96 well plate, and the volume of the staining solution can be adjusted according to experimental needs. The concentrations of Calcein AM and EthD-I can range from 0.1 to 10 μ Explore between M.
(2) The aqueous solution of Calcein AM is easily hydrolyzed and should be used up every day. EthD-I working solution can be stored at -20 ℃ for at least one year.
3. Wash the cells thoroughly with 1 × PBS to remove residual esterase activity. For adherent cells, add 100 to each well μ Wash cells with PBS. For suspended cells, add 100 μ Resuspend cells with L PBS and centrifuge to remove the supernatant. Repeat the above operation.
4. Add 100 to each hole μ L PBS.
5. Add 100 to each hole μ L staining solution, making the total volume of each well 200 μ L. The final concentration of Calcein AM is 1 μ M. The final concentration of EthD-I is 2 μ M. Gently shake the culture plate to evenly cover the cells with the liquid.
Incubate at room temperature in dark for 30-45 minutes.
Note: The optimal incubation time varies for different cells, with 30 minutes as the initial incubation time. Subsequently, the staining time can be adjusted and optimized according to the actual staining effect to obtain a more ideal staining effect.
7. Enzyme reader detection. When the ELISA reader is set to fluorescein, it can detect Calcein AM; When the ELISA reader is set to rhodamine or Texas Red, EthD-I can be detected. Select the optimal emission and excitation wavelengths based on spectral characteristics.
Note: By comparing the relative fluorescence values (RFU) measured between the sample group and the control group, the changes in the number of dead and live cells can be obtained. Another method of data analysis is also provided below.
The following method can calculate the ratio of live cells to dead cells in a certain region. The required samples include dead cell control group, live cell control group, and the sample group to be tested. Dead cells can be obtained by treating cells with 1% saponin or 0.1-0.5% digitalis saponin for 10 minutes.
1. Prepare staining solution and follow the above steps to stain cells. Additionally, prepare 1 mL and 2 mL separately μ M Calcein AM and 4 μ M EthD-I solution, stain the control group according to the following instructions. For the following groups of cells or cell-free groups, it is necessary to maintain complete consistency in cell count, detection of working solution concentration, incubation time, and incubation temperature.
2. Measurement of sample group and control group:
A. The measured values of the sample group at 645 nm are denoted as Calcein AM and EthD-I=F (645) sam.
B. The measured values of the sample group at 530 nm are denoted as Calcein AM and EthD-I=F (530) sam.
C. The measurement value of dead cell EthD-I single staining control group at 645 nm is denoted as EthD-I=F (645) max
D. The measurement value of dead cell Calcein AM single staining control group at 645 nm is recorded as Calcein AM=F (645) min
E. The measurement value of live cell EthD-I single staining control group at 530 nm is recorded as EthD-I=F (530) min.
F. The measurement value of live cell Calcein AM single staining control group at 530 nm is denoted as Calcein AM=F (530) max.
G. A blank control well without cells (with or without dye), the detection value at 530 nm is recorded as F (530) 0.
H. A blank control well without cells (with or without dye), the detection value at 645 nm is recorded as F (645) 0.
3. Calculate the ratio of dead cells to live cells based on measurement data:
%Live Cells=(B-E) ÷ (F-E)
%Dead Cells=(A-D) ÷ (C-D)
Determine the ratio of live cells to dead cells in a certain area
By creating fluorescence spectral standard curves at 530 nm and 645 nm, the number of dead and live cells can be determined, and the fluorescence intensity of each dye is linearly related to the number of dead or live cells in the sample.
Matters needing attention：
1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments.
2. phenol red or serum may interfere with the detection of this kit.
3. fluorescent dyes have quenching problems. Please try to avoid light during experimental operation to slow down fluorescence quenching.
4. for your safety and health, please wear experimental clothes and disposable gloves.
Scope of application：
Dead and live cell staining (animal)
规格
英文名称
Live & deadtm animal cell viability / toxicity detection kit (calcein am, ethd-1)
备注
注意Calcein AM容易水解，需密封干燥保存，稀释工作液需当天配制。
储存与运输
储存条件
避光,-20°C储存
运输条件
超低温运输
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
技术文档和文章
此产品的引用文献
溶液计算器
产品仅提供给经过验证的企业、机构和合格的专业人员用于研发。不适用于人类、动物、诊断或治疗。
Copyright © 2023至今 上海阿拉丁生化科技股份有限公司 保留所有权利。
危险品化学品经营许可证（带存储）