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CFDA SE细胞增殖与示踪检测试剂盒(CFDA SE Cell Proliferation Assay and Tracking Kit)，是基于一种新型荧光探针CFDA SE的用于细胞增殖检测和细胞荧光示踪的检测试剂盒。CFDA SE目前被广泛用于替代MTT法或[3H]-thymidine掺入等其它细胞增殖检测方法。
CFDA-SE的全称为Carboxyfluorescein diacetate, succinimidyl ester，分子式为C29H19NO11，分子量为557.47，CAS number为150347-59-4。CFDA SE可以通透细胞膜，进入细胞后可以被细胞内的酯酶(esterase)催化分解成CFSE，CFSE可以偶发性地(spontaneously)并不可逆地和细胞内蛋白的Lysine残基或其它氨基发生结合反应，并标记这些蛋白。在加入荧光探针CFDA SE后大约24小时，即可充分标记细胞。
使用方法
1. 检测前的准备：
a.加入500 μL CFDA,SE溶剂到1管CFDA,SE荧光探针中进行溶解，充分混匀即得到1000×的储存液。该储存液最好在1个月内使用完毕，最长不超过2个月。剩余储存液请务必分装后于≤-20℃避光干燥保存，避免反复冻融，-70℃避光保存可适当延长保存周期。
b. CFDA SE细胞标记液的配制：准备适量无菌的细胞培养级纯水，根据实验需要配制适量的CFDA SE细胞标记液。例如，取20ml CFDA SE细胞标记液(10X)，加入180ml细胞培养级纯水，混匀后即为CFDA SE细胞标记液。配制好的CFDA SE细胞标记液可以4ºC保存，长时间不用可以-20ºC保存。注意：虽然本试剂盒已对CFDA,SE染色体系做了优化处理，但建议使用者根据自身的细胞类型，培养条件以及应用的不同来梯度摸索最佳的工作浓度，以最低工作浓度得到适宜的标记效率为准。
2. 操作步骤
1） 离心收集细胞，利用1mL 1×细胞染色缓冲液悬浮细胞于15 mL离心管，并调整细胞浓度为1-5×106个/mL；
2） 把1 mL CFDA,SE工作液(2×)加入到上述15 mL离心管内，轻轻混匀。
3） 37℃孵育10 min。注意：对于不同细胞，需要自行摸索最佳标记时间。
4） 立即在15 mL离心管内加入约10 mL完全细胞培养液(含10% FBS)，室温颠倒数下混匀，以终止标记反应。
5） 室温离心去上清，再用5-10 mL完全细胞培养液洗涤一次。
6） 再加入5-10 mL完全细胞培养液，37℃孵育10 min，以促进CFSE酯酶催化CFDA,SE得到的荧光产物在细胞内的驻留及未反应的CFDA,SE进入完全细胞培养液。离心去上清，完成最后一次洗涤。
7） 此时标记好的细胞已可进行后续的体外增殖检测或者特定目的的细胞示踪。按照正常方法培养细胞，然后在合适的时间点用荧光显微镜或流式细胞仪FL1通道进行结果分析，呈绿色荧光。标记的细胞也可用于活体动物的移植，并用荧光进行示踪。
注意：若需要对细胞进行固定，请用醛类固定剂如3.7%多聚甲醛于室温固定15 min；之后若还需要进行其他如抗体标记，请用冰丙酮透化处理细胞10 min。
注意事项：
1. CFDA SE配制成储存液后宜在一个月内使用完毕，最长不宜超过2个月。CFDA SE易被水解，在水溶液中会很快变质。请在使用过程中避免接触水。在标记细胞的过程中和水接触是在许可的范围内的。
2. CFDA SE溶剂在4ºC、冰浴等较低温度情况下会凝固而粘在离心管管底、管壁或管盖内，可以20-25ºC水浴温育片刻至全部融解后使用。
3. 荧光染料均存在淬灭问题，请尽量注意避光，以减缓荧光淬灭。
4. 本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。
5. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
CFDA SE Cell Proliferation Assay and Tracking Kit is a detection kit for cell proliferation assay and cell fluorescence tracking based on a novel fluorescent probe, CFDA SE.CFDA SE is now widely used as an alternative to the MTT assay, [3H]-thymidine incorporation, and other cell proliferation detection methods.
The full name of CFDA‑SE is Carboxyfluorescein diacetate, succinimidyl ester, with a molecular formula of C₂₉H₁₉NO₁₁, a molecular weight of 557.47, and a CAS number of 150347-59-4.CFDA SE is cell‑membrane permeable.After entering cells, it is catalyzed and hydrolyzed by intracellular esterase into CFSE.CFSE can spontaneously and irreversibly bind to lysine residues or other amino groups of intracellular proteins and label these proteins.
Cells can be sufficiently labeled approximately 24 hours after the addition of the fluorescent probe CFDA SE.
Instructions
1. Preassay Preparation
a. Add 500 μL of CFDA SE Solvent to one tube of CFDA SE fluorescent probe, dissolve and mix thoroughly to obtain 1000× stock solution.The stock solution is best used within 1 month, and for a maximum of 2 months.Aliquot any remaining stock solution and store at ≤ −20°C, protected from light and dry, avoiding repeated freeze‑thaw cycles.Storage at −70°C protected from light may appropriately extend the shelf life.
b. Preparation of CFDA SE Cell Labeling Solution:
Prepare an appropriate amount of sterile cell culturegrade water.Prepare sufficient CFDA SE Cell Labeling Solution according to experimental needs.For example: take 20 mL of 10× CFDA SE Cell Labeling Solution, add 180 mL of cell culture‑grade water, and mix well to obtain 1× working solution.The prepared CFDA SE Cell Labeling Solution can be stored at 4°C, or at −20°C for long‑term storage.
Note: Although the kit has optimized the CFDA SE staining system, users are recommended to optimize the working concentration gradient according to their own cell type, culture conditions and application, using the lowest working concentration that achieves satisfactory labeling efficiency.
2. Procedure
1) Harvest cells by centrifugation.
Suspend cells in 1 mL of 1× Cell Staining Buffer in a 15 mL centrifuge tube, and adjust the cell concentration to 1–5×10⁶ cells/mL.
2) Add 1 mL of 2× CFDA SE working solution to the above 15 mL centrifuge tube and mix gently.
3) Incubate at 37°C for 10 min.Note: The optimal labeling time should be optimized for different cell types.
4) Immediately add approximately 10 mL of complete cell culture medium (containing 10% FBS) to the 15 mL centrifuge tube.Invert gently several times at room temperature to stop the labeling reaction.
5) Centrifuge at room temperature and discard the supernatant.Wash once with 5–10 mL of complete cell culture medium.
6) Add another 5–10 mL of complete cell culture medium and incubate at 37°C for 10 minto promote intracellular retention of the fluorescent product formed from CFDA SE by esterase catalysis,and to allow unreacted CFDA SE to diffuse into the medium.Centrifuge and discard the supernatant to complete the final wash.
7) The labeled cells are now ready for subsequent in vitro proliferation assays or cell tracking for specific purposes.Culture cells under normal conditions, then analyze at appropriate time points using a fluorescence microscope or flow cytometer FL1 channel.Labeled cells show green fluorescence.Labeled cells can also be transplanted into living animals and traced by fluorescence.Note:If cell fixation is required, fix cells with an aldehyde fixative such as 3.7% paraformaldehyde at room temperature for 15 min.If further staining (e.g., antibody labeling) is needed afterward, permeabilize cells with ice‑cold acetone for 10 min.
Precautions
1.CFDA SE is susceptible to hydrolysis and degrades rapidly in aqueous solution.Avoid contact with water during handling, except during cell labeling, which is permitted.
2.The CFDA SE Solvent may solidify at low temperatures such as 4°C or on ice, adhering to the bottom, wall or cap of the centrifuge tube.Incubate in a water bath at 20–25°C briefly until fully dissolved before use.
3.All fluorescent dyes are prone to photobleaching.Protect from light as much as possible to slow down fluorescence quenching.
4.This product is for research use only by professional personnel.It is not intended for clinical diagnosis or therapy,not for use in food or pharmaceutical products,and must not be stored in ordinary residential premises.
5. For your safety and health,please wear a lab coat and disposable gloves during operation.
C1507077 | Components | 500T | 1000T | 2000T | 储存 |
C1507077A | CFDA SE荧光探针 | 1管 | 2管 | 4管 | -20℃, 避光保存 |
C1507077B | CFDA SE溶剂 | 1 mL | 2 x 1 mL | 4 x 1 mL | -20℃, 避光保存 |
C1507077C | 细胞染色缓冲液 | 50 mL | 100 mL | 200 mL | -20℃. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0433908 |
分析证书 | C1507077 |
| ZJ26F0433907 |
分析证书 | C1507077 |
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