J2528183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 0 篇同行评审文献引用。
概述
细胞增殖检测是评估细胞健康程度、遗传毒性及抗肿瘤药物效果的基础实验手段。检测细胞增殖最精确的方法是 BrdU法。EdU法检测试剂盒是BrdU法的革命性突破。EdU（5-乙炔基-2’-脱氧尿苷）是一种嘧啶类似物，在DNA合成期整合入DNA双链。EdU法检测基于“点击”反应，一种由铜催化的叠氮化合物和炔烃作用发生共价反应，形成共价键。 本试剂盒中，EdU含有炔烃，aladdin® 488/555/594/647A Azide染料含有叠氮化合物。点击法的EdU标记增殖快速有效，易于使用。BrdU方法需要DNA变性（如酸变性、热变性或者用DNase消化）暴露出BrdU，从而方便BrdU抗体结合；而EdU法只需多聚甲醛固定和Triton X-100促渗就可以使检测试剂进入细胞，只需少量的叠氮化染料即可非常有效地标记出整合的EdU。 本试剂盒包含EdU法检测所需要的所有组分，可以用于体外培养细胞的增殖检测。
组分：
产品参数：
590/617 nm
使用方法：
实验材料（自备）
.10 mM PBS, pH 7.2-7.6
.4 %多聚甲醛固定液（in PBS）
.促渗试剂（0.5 %Triton X- 100 in PBS）
.2 mg/mL 甘氨酸溶液（in ddH2O）
.3 %BSA in PBS, pH 7.2-7.6
.1 % BSA in PBS, pH 7.2-7.6
.ddH2O
.96/24/12/6 孔培养板或培养皿
荧光显微镜检测方法
1. 细胞培养
取对数生长期细胞， 以每孔4×103- 1×105细胞（可根据细胞大小、生长速度以及实验处理的具体要求调整细胞数量和密度）接种于96孔板中，培养至正常生长阶段。
2. 药物处理
根据实验需要进行各种药物处理。
3. EdU标记
（1）用细胞完全培养基按一定比例稀释 EdU 溶液（组分 A）至合适浓度后加入细胞中，混匀；设置不加 EdU 处理的阴性对照组。
注： EdU 的标记浓度需根据细胞类型加以调整，建议以 10 µM 的初始浓度进行摸索。预实验中，建议设置 EdU 浓度梯度，可参考附表 2 和附表 3。
（2）细胞培养箱中孵育 2 h。
注：最佳孵育时间与细胞周期有关， 大多数肿瘤细胞系均可采用 2 h 的孵育时间， 可参考附表 2。EdU 浓度与孵育时间相关，
短时间孵育（<2 h）宜采用高浓度，如： 10~50 μM；长时间孵育（>24 h）宜采用低浓度，如： 1~10 μM；也可参考附表 3。
4. 细胞固定及促渗
注： 对于需要做细胞表面抗原标记的实验， 可以考虑在完成 EdU 孵育后， 以含 3% BSA 洗涤液洗涤细胞 2 次， 在细胞固定促渗之前进行。
（1）孵育完成后， 去除培养基。 以 1X PBS 清洗细胞两次，每次5 min，以除去未掺入 DNA 的 EdU 残留， 贴壁不牢的细胞可降低清洗强度。 加入50 μL 4 % 多聚甲醛固定液，室温孵育20 min后，去除固定液。
（2）每孔加入50 μL 2 mg/mL甘氨酸溶液，室温孵育5 min，中和残留的固定液。
（3）以每孔100 μL 3 % BSA洗涤细胞2次。
（4）去除洗涤液，加入100 μL 0.5 % Triton X- 100，室温孵育10 min。
5. EdU检测
注：本参考步骤每个样本使用100 μL的工作液， 用户可以根据自己的样本情况调整用量。
（1）配置1×Click-iT EdU反应缓冲液（组分C）：用ddH2O将组分C稀释10倍。
（2）配置5×Click-iT EdU缓冲液添加物（组分E）：加300 μL的ddH2O至30 mg的E组分管中（终浓度100 mg/mL），混匀至全部溶解。使用后，剩余储液存放在-20℃,可保存一年，溶液一旦呈现棕色，则说明有效成分降解不能再用。
注：不同规格的组分E均按照此比例加ddH2O溶解，制备成5× 储液备用。
（3）准备1×Click-iT EdU缓冲液添加物：用ddH2O稀释5×Click-iT EdU缓冲液添加物至1×,溶液应现配现用。
（4）依据表 1 准备 Click-iT 工作液。
表 1. Click-iT 工作液
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（5）去除促渗剂，每孔100 μL的3% BSA洗涤液洗涤2次。
（6）每孔加入100 μL Click-iT工作液，均匀覆盖细胞。
（7）室温避光孵育 30 min。
（8）除去 Click-iT 工作液，以 100 μL 3% BSA 洗涤细胞 2 次后，去除洗涤液, 加入 100 μL PBS 保持细胞湿润。如其他无特别要求，即可进行拍照分析。
6. DNA 复染（可选）
（1）用 100 μL PBS 洗涤细胞 1 次，去除洗涤液。
（2）用 PBS 将 Hoechst 33342 （组分 F）稀释 2000 倍。
（3）每孔加 100 μL 1×Hoechst 33342 溶液，室温避光孵育 15-30 min。
（4）去除 Hoechst 33342 溶液，用 100 μL PBS 洗涤细胞 2 次。
7. 成像及分析
建议染色完成后立即进行荧光显微镜拍照观察；如果条件限制，请于 4°C 条件下避光湿润保存 3 天之内完成拍照。流式细胞仪检测方法
1. 细胞培养
每孔 1×105~3×106 个细胞接种于6孔板中。
2. 药物处理
根据实验需要进行各种药物处理。
3. EdU 标记细胞
（1）用细胞完全培养基按一定比例稀释 EdU 溶液（组分 A）至合适浓度后加入细胞中，混匀；设置不加 EdU 处理的阴性对照组。
注： EdU 的标记浓度需根据细胞类型加以调整，建议以 10 µM 的初始浓度进行摸索。预实验中，建议设置 EdU 浓度梯度，可参考附表 2 和附表 3。
（2）细胞培养箱中孵育 2 h 。EdU 孵育细胞的时间可以直接用作测定细胞 DNA 合成的指标， 时间点选择以及孵育的时间取决于细胞生长速率。通过短暂的 EdU 孵育进行的脉冲式标记细胞可以用于研究细胞周期动力学。
注：最佳孵育时间与细胞周期有关， 大多数肿瘤细胞系均可采用 2 h 的孵育时间， 可参考附表 2。EdU 浓度与孵育时间相关， 短时间孵育（<2 h）宜采用高浓度，如： 10~50 μM；长时间孵育（>24 h）宜采用低浓度，如： 1~10 μM；也可参考附表 3。
4. 细胞固定及促渗
注：对于需要做细胞表面抗原标记的实验，可以考虑在完成 EdU 孵育后，以含 1 % BSA 洗涤细胞 2 次，在细胞固定促渗之前进行。
（1）孵育完成后，收集细胞，每管加入1 mL PBS 清洗细胞， 1000 rpm离心5 min，吸弃上清，以除去未掺入 DNA的 EdU残留。
（2）每管加入 1 mL 4 % 多聚甲醛固定液重悬细胞。
（3）室温孵育 20 min ，1000 rpm 离心 5 min ，吸弃上清。
（4）每管加入 1 mL 2 mg/mL 甘氨酸孵育 5min，中和残留的固定液， 1000 rpm 离心 5 min，吸弃上清，每管加入 1 mL PBS清洗 1 次， 1000 rpm 离心 5 min，吸弃上清。
（5）每管加入 1mL 0.5 % Triton X- 100 促渗液重悬细胞，室温孵育 10 min。
5. EdU 检测
注： 针对 6 孔板样本可参考每孔 1 mL 的工作液来进行，用户可以根据自己的样本情况调整用量。
（1）配置1×Click-iT EdU反应缓冲液：用ddH2O将组分C稀释10倍。
（2）配置 5×Click-iT EdU 缓冲液添加物（组分 E）：加 300 µL ddH2O 至 30 mg 的组分 E 试管中（终浓度 100 mg/mL），混匀至全部溶解。使用后，剩余储液存放在-20℃,可保存一年，溶液一旦呈现棕色，则说明有效成分降解不能再用。
注：不同规格的组分 E 均按照此比例加 ddH2O 溶解为 5× 储液备用。
（3）准备1×Click-iT EdU缓冲液添加物：以ddH2O稀释5×Click-iT EdU缓冲液添加物储液至1×,溶液应现配现用。
（4）依据表 2 准备 Click-iT 工作液。
表 2. Click-iT 工作液
|
（5）1000 rpm 离 5 min，吸弃上清，去除促渗剂，每管加入 1mL 的 1% BSA 洗涤液洗涤 2 次， 1000 rpm 离 5 min，吸弃上清。
（6）每管加入 1 mL Click-iT 工作液，混匀。
（7）室温避光孵育 30 min。
（8）1000 rpm 离 5 min，吸弃染色反应液，每管加入 1% BSA 洗涤细胞 2 次， 1000 rpm 离心 5min，吸弃上清，用 1 mL 1 %BSA 再次重悬细胞（重悬细胞的溶液体积可根据细胞的数量加以调整），流式细胞仪检测。
注：如需进行其他标志物检测可参考步骤 4。
6. 细胞内抗原标记（可选步骤）
（1）加入抗体工作液，混匀。
（2）避光条件下，以合适的温度及时间孵育抗体。
7. 流式检测及分析:
（1）建议染色完成后立即进行流式检测；如果条件限制，请避光 4℃湿润保存待测，但不应超过 3 天。
（2）检测的细胞数量建议尽量能达到百万级，若细胞数量较少，检测的细胞数量可调整为十万级起始进行实验。对于细胞得率过少（刚到万级）的情况，可能不利于做流式图，对此可适当减少步骤 5（8）中的清洗次数。
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. 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。
3. Click-iT EdU缓冲液添加物溶液最好现配现用，以保证最佳结果。
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：
细胞增殖检测（细胞成像流式通用款）
Cell proliferation detection is a basic experimental method to evaluate the health of cells, genotoxicity and the effect of antitumor drugs. The most accurate method to detect cell proliferation is the BrdU method. Edu detection kit is a revolutionary breakthrough of BrdU method. Edu (5-ethynyl-2 '- deoxyuridine) is a pyrimidine analog that integrates into the DNA duplex during DNA synthesis. Edu detection is based on the "click" reaction. A copper catalyzed azide reacts covalently with alkynes to form covalent bonds. In this kit, edu contains alkynes, Aladdin ® 488 / 555/594/647a azide dyes contain azide compounds. The edu labeling proliferation of click method is rapid and effective, and easy to use. BrdU method requires DNA denaturation (such as acid denaturation, thermal denaturation or digestion with DNase) to expose BrdU, so as to facilitate BrdU antibody binding; The edu method only needs paraformaldehyde fixation and Triton X-100 penetration to make the detection reagent enter the cells, and only a small amount of azide dye is needed to label the integrated edu very effectively. This kit contains all components required for edu method detection, and can be used for proliferation detection of cultured cells in vitro.
Component：
Product parameters：
590/617 nm
Instruction：
Experimental materials (self provided)
. 10 mM PBS, pH 7.2-7.6
. 4% paraformaldehyde fixing solution (in PBS)
Propensive reagent (0.5% Triton X -100 in PBS)
. 2 mg/mL glycine solution (in ddH2O)
. 3% BSA in PBS, pH 7.2-7.6
. 1% BSA in PBS, pH 7.2-7.6
. ddH2O
. 96/24/12/6 well culture plate or dish
Fluorescence microscopy detection method
1. Cell culture
Take logarithmic growth stage cells and inoculate them into a 96 well plate with 4 × 103-1 × 105 cells per well (the number and density of cells can be adjusted according to cell size, growth rate, and specific requirements of experimental treatment), and culture until normal growth stage.
2. Drug treatment
Perform various drug treatments according to experimental needs.
3. EdU marking
(1) Dilute EdU solution (component A) in a certain proportion with complete cell culture medium to an appropriate concentration, then add it to the cells and mix well; Set up a negative control group without EdU treatment.
Note: The labeling concentration of EdU needs to be adjusted according to cell type, and it is recommended to explore with an initial concentration of 10 µ M. In the pre experiment, it is recommended to set an EdU concentration gradient, which can be referred to in Tables 2 and 3.
(2) Incubate in a cell culture incubator for 2 hours.
Note: The optimal incubation time is related to the cell cycle. Most tumor cell lines can use a 2-hour incubation time, as shown in Appendix 2. The concentration of EdU is related to incubation time,
Short term incubation (<2 hours) should use high concentrations, such as 10-50 μ M; Long term incubation (>24 hours) should use low concentrations, such as 1-10 μ M; You can also refer to Appendix 3.
4. Cell fixation and permeation promotion
Note: For experiments that require cell surface antigen labeling, it can be considered to wash the cells twice with a 3% BSA washing solution after completing EdU incubation, before cell fixation and permeation promotion.
(1) After incubation, remove the culture medium. Wash cells twice with 1X PBS for 5 minutes each time to remove EdU residues that have not been incorporated into DNA. Cells with weak adhesion can reduce cleaning intensity. Join 50 μ After incubating at room temperature for 20 minutes with 4% paraformaldehyde fixative, remove the fixative.
(2) Add 50 to each hole μ L 2 mg/mL glycine solution, incubate at room temperature for 5 minutes, and neutralize the remaining fixed solution.
(3) At a rate of 100 per hole μ Wash cells twice with 3% BSA.
(4) Remove the washing solution and add 100 μ L 0.5% Triton X -100, incubate at room temperature for 10 minutes.
5. EdU detection
Note: Each sample in this reference step uses 100 μ The working fluid of L can be adjusted by users according to their own sample situation.
(1) Prepare 1 x Click iT EdU reaction buffer (component C): Dilute component C 10 times with ddH2O.
(2) Configure 5 x Click iT EdU buffer additives (component E): add 300 μ Mix L of ddH2O into a 30 mg E component tube (final concentration of 100 mg/mL) until completely dissolved. After use, the remaining storage solution is stored at -20 ℃ and can be stored for one year. Once the solution turns brown, it indicates that the active ingredients have degraded and cannot be reused.
Note: Different specifications of component E are dissolved in ddH2O according to this ratio, and prepared into a 5 x storage solution for future use.
(3) Prepare 1 x Click iT EdU buffer additive: Dilute 5 x Click iT EdU buffer additive with ddH2O to 1 x, and the solution should be prepared and used immediately.
(4) Prepare Click it working solution according to Table 1.
Table 1 Click it working fluid
|
(5) Remove penetration enhancer, 100 per well μ Wash twice with 3% BSA washing solution of L.
(6) Add 100 to each hole μ L Click iT working solution, evenly covering cells.
(7) Incubate at room temperature in dark for 30 minutes.
(8) Remove Click-iT working fluid and add 100 μ After washing cells twice with 3% BSA, remove the washing solution and add 100 μ L PBS keeps cells moist. If there are no other special requirements, photography analysis can be carried out.
6. DNA re staining (optional)
(1) Using 100 μ Wash the cells once with PBS and remove the washing solution.
(2) Dilute Hoechst 33342 (component F) 2000 times with PBS.
(3) Add 100 to each hole μ Incubate L 1 x Hoechst 33342 solution at room temperature in dark for 15-30 minutes.
(4) Remove Hoechst 33342 solution and use 100 μ Wash cells twice with PBS.
7. Imaging and analysis
It is recommended to take fluorescence microscopy photos immediately after staining is completed for observation; If conditions permit, please store in a dark and moist environment at 4 ° C for 3 days before taking photos. Flow cytometry detection method
1. Cell culture
Inoculate 1 × 105~3 × 106 cells per well into a 6-well plate.
2. Drug treatment
Perform various drug treatments according to experimental needs.
3. EdU labeled cells
(1) Dilute EdU solution (component A) in a certain proportion with complete cell culture medium to an appropriate concentration, then add it to the cells and mix well; Set up a negative control group without EdU treatment.
Note: The labeling concentration of EdU needs to be adjusted according to cell type, and it is recommended to explore with an initial concentration of 10 µ M. In the pre experiment, it is recommended to set an EdU concentration gradient, which can be referred to in Tables 2 and 3.
(2) Incubate in a cell culture incubator for 2 hours. The time of EdU incubation of cells can be directly used as an indicator for measuring cell DNA synthesis, and the choice of time point and incubation time depend on the cell growth rate. Pulse labeled cells incubated with brief EdU can be used to study cell cycle dynamics.
Note: The optimal incubation time is related to the cell cycle. Most tumor cell lines can use a 2-hour incubation time, as shown in Appendix 2. The concentration of EdU is related to the incubation time, and high concentrations, such as 10-50, should be used for short-term incubation (<2 hours) μ M; Long term incubation (>24 hours) should use low concentrations, such as 1-10 μ M; You can also refer to Appendix 3.
4. Cell fixation and permeation promotion
Note: For experiments that require cell surface antigen labeling, it can be considered to wash cells twice with 1% BSA after completing EdU incubation, before cell fixation and permeation promotion.
(1) After incubation, collect cells, add 1 mL of PBS to each tube to clean the cells, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant to remove EdU residue that has not been added to DNA.
(2) Add 1 mL of 4% paraformaldehyde fixative to each tube to resuspend cells.
(3) Incubate at room temperature for 20 minutes, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant.
(4) Add 1 mL of 2 mg/mL glycine to each tube and incubate for 5 minutes. Neutralize the remaining fixed solution, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Add 1 mL of PBS to each tube for cleaning once, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant.
(5) Add 1mL of 0.5% Triton X-100 osmotic enhancer to each tube and resuspend cells. Incubate at room temperature for 10 minutes.
5. EdU detection
Note: For 6-well plate samples, reference can be made to 1 mL of working solution per well. Users can adjust the dosage according to their own sample situation.
(1) Prepare 1 x Click iT EdU reaction buffer: Dilute component C 10 times with ddH2O.
(2) Prepare 5 x Click iT EdU buffer additives (component E): Add 300 µ L ddH2O to 30 mg of component E in a test tube (final concentration 100 mg/mL), mix well until completely dissolved. After use, the remaining storage solution is stored at -20 ℃ and can be stored for one year. Once the solution turns brown, it indicates that the active ingredients have degraded and cannot be reused.
Note: Different specifications of component E are dissolved in ddH2O according to this ratio to form 5 x storage solution for future use.
(3) Prepare 1 x Click iT EdU buffer additive: Dilute 5 x Click iT EdU buffer additive storage solution with ddH2O to 1 x, and the solution should be prepared and used immediately.
(4) Prepare Click it working solution according to Table 2.
Table 2 Click it working fluid
|
(5) Soak at 1000 rpm for 5 minutes, discard the supernatant, remove the enhancer, add 1mL of 1% BSA washing solution to each tube and wash twice. Soak at 1000 rpm for 5 minutes, discard the supernatant.
(6) Add 1 mL of Click iT working solution to each tube and mix well.
(7) Incubate at room temperature in dark for 30 minutes.
(8) Soak at 1000 rpm for 5 minutes, discard the staining reaction solution, add 1% BSA to each tube to wash the cells twice, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend the cells again with 1 mL of 1% BSA (the volume of resuspend cells can be adjusted according to the number of cells), and detect with a flow cytometer.
Note: If other biomarker tests are required, please refer to step 4.
6. Intracellular antigen labeling (optional steps)
(1) Add antibody working solution and mix well.
(2) Under dark conditions, incubate antibodies at appropriate temperature and time.
7. Flow detection and analysis:
(1) It is recommended to conduct flow cytometry testing immediately after dyeing is completed; If conditions are limited, please store in a dark place at 4 ℃ for testing, but it should not exceed 3 days.
(2) It is recommended to test the number of cells up to one million levels as much as possible. If the number of cells is small, the number of cells tested can be adjusted to 100000 levels starting from the experiment. For cases where the cell yield is too low (just to the level of ten thousand), it may not be conducive to making a flow chart. Therefore, the cleaning frequency in step 5 (8) can be appropriately reduced.
Matters needing attention：
1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments.
2. fluorescent dyes have quenching problems. Please try to avoid light during experimental operation to slow down fluorescence quenching.
3. click it edu buffer additive solution should be prepared and used immediately to ensure the best results.
4. for your safety and health, please wear experimental clothes and disposable gloves.
Scope of application：
Cell proliferation detection (cell imaging flow universal)
规格
英文名称
Aladdin ® 594 click it edu universal cell proliferation detection kit (red fluorescence)
备注
本产品即将售完下架，替代产品为E1373492
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
溶液计算器
常见问题
本产品应如何储存？
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