Fluorescent Dye

5-CFDA溶液配制方法
一、母液制备步骤
1. 取适量5-CFDA粉末溶于DMSO溶剂中，配制成浓度为10 mM的储备液。具体操作示例：将1毫克5-CFDA溶解于0.2172毫升DMSO中。
重要提示：配制好的储备液需分装保存于-20℃或-80℃低温环境中，并注意避光存放。
二、工作液配制流程
1. 使用预热的无血清培养基或PBS缓冲液对储备液进行稀释，最终浓度控制在1-10 μM范围。
特别说明：工作液的具体使用浓度需根据实验需求进行调整，且必须现配现用。
细胞标记实验流程
1. 样本前处理
• 悬浮细胞系：通过离心收集细胞后，使用PBS缓冲液清洗两次，每次清洗时间5分钟。
• 贴壁细胞系：移除培养液后加入胰酶消化，离心去除上清液，同样用PBS清洗两次，每次5分钟。
2. 染色过程
• 加入1毫升配制好的5-CFDA工作液，在室温条件下孵育15分钟。
3. 后处理步骤
• 4℃环境下以400g离心力离心3-4分钟，去除上清液。
• 再次使用PBS缓冲液清洗细胞两次，每次5分钟。
• 最后用1毫升无血清培养基或PBS重悬细胞，即可进行荧光显微镜观察或流式细胞仪检测。

实验注意事项
1. 储存条件：5-CFDA储备液必须分装保存于-20℃（有效期1个月）或-80℃（有效期6个月），注意避光并避免反复冻融。
2. 浓度调整：工作液使用浓度应根据具体实验条件进行优化。
3. 使用限制：本试剂仅供科研使用，禁止用于临床诊疗、食品或药品领域。
4. 安全防护：实验操作过程中需穿着实验服并佩戴一次性手套，确保实验人员安全。

[1]. A novel nonradioactive CFDA assay to monitor the cellular immune response in myeloid leukemia. Immunobiology. 2013 Apr;218(4):548-53.

[2]. Assessment of fluorescein-based fluorescent dyes for tracing Neotyphodium endophytes in planta. Mycologia. 2013 Jan-Feb;105(1):221-9.

[3]. Bone marrow-derived endothelial progenitor cells are involved in aneurysm repair in rabbits. J Clin Neurosci. 2012 Sep;19(9):1283-6.

Endothelial progenitor cells (EPC) are believed to be involved in aneurysmal repair and remodeling. The aim of this study was to test this hypothesis and, if true, explore how EPC contribute to aneurysm repair in a rabbit model of elastase-induced carotid aneurysm. Rabbits were divided randomly into an in situ carotid EPC transfusion group (ISCT group, n=5), and an intravenous EPC transfusion group (IVT group, n=5). Autologous EPC were double-labeled with Hoechst 33342 and 5,6-carboxyfluorescein diacetate succinimidyl ester before injection into the animals in either the carotid artery (ISCT group) or marginal ear veins (IVT group). Three weeks later, labeled cells in the aneurysms were observed with respect to location, adhesion, and growth to detect signs of aneurysm repair. Labeled EPC were detected within the neointima in all five aneurysms in the ISCT group and in three of the five aneurysms in the IVT group, but there was no endothelial growth in the aneurysmal neointima in either group. These results show that bone marrow-derived EPC are involved in the process of aneurysm repair in this rabbit model. [1]
Fluorescent dyes were assessed for their ability to stain viable hyphae of the fungi Neotyphodium lolii and N. coenophialum, symbiotic endophytes of the Pooideae grasses Lolium perenne and Festuca arundinacea, respectively. The fluorescein-based fluorophores; fluorescein diacetate (FDA), 5(6)-carboxy-fluorescein diacetate (CFDA), 5-chloromethylfluorescein diacetate (CMFDA) and the chitin-binding stain, Calcofluor while M2R, were assessed for staining of endophyte hyphae in vitro from axenic fungal cultures and in planta, including epidermal leaf sheath peels, nodes, ovaries, embryos and meristems. CMFDA produced the greatest intensity of staining of fungal hyphae and gave excellent contrast in planta compared to the plant cells. Compared to the other dyes, CMFDA was also the least affected by photo bleaching and continued to fluoresce up to 2 h after initial excitation. None of the fluorescent dyes stained fungal hyphae in seed. [2]

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Endothelial progenitor cells (EPC) are believed to be involved in aneurysmal repair and remodeling. The aim of this study was to test this hypothesis and, if true, explore how EPC contribute to aneurysm repair in a rabbit model of elastase-induced carotid aneurysm. Rabbits were divided randomly into an in situ carotid EPC transfusion group (ISCT group, n=5), and an intravenous EPC transfusion group (IVT group, n=5). Autologous EPC were double-labeled with Hoechst 33342 and 5,6-carboxyfluorescein diacetate succinimidyl ester before injection into the animals in either the carotid artery (ISCT group) or marginal ear veins (IVT group). Three weeks later, labeled cells in the aneurysms were observed with respect to location, adhesion, and growth to detect signs of aneurysm repair. Labeled EPC were detected within the neointima in all five aneurysms in the ISCT group and in three of the five aneurysms in the IVT group, but there was no endothelial growth in the aneurysmal neointima in either group. These results show that bone marrow-derived EPC are involved in the process of aneurysm repair in this rabbit model. [3]

C25H16O9

460.389147758484

460.079

C, 65.22; H, 3.50; O, 31.28

79955-27-4

6-CFDA;3348-03-6;5(6)-CFDA;124387-19-5

133314

Off-white to yellow solid powder

1.6±0.1 g/cm3

693.1±55.0 °C at 760 mmHg

241.9±25.0 °C

0.0±2.3 mmHg at 25°C

1.702

2.4

125.43

1

9

5

34

828

0

O=C(C1C=C2C(OC3(C4C(=CC(=CC=4)OC(C)=O)OC4C3=CC=C(C=4)OC(C)=O)C2=CC=1)=O)O

WPUZGNPQMIWOHE-UHFFFAOYSA-N

InChI=1S/C25H16O9/c1-12(26)31-15-4-7-19-21(10-15)33-22-11-16(32-13(2)27)5-8-20(22)25(19)18-6-3-14(23(28)29)9-17(18)24(30)34-25/h3-11H,1-2H3,(H,28,29)

3',6'-Diacetyloxy-3-oxospiro[2-benzofuran-1,9'-xanthene]-5-carboxylic acid

5CFDA; 5-CFDA; 5 CFDA; 5-Carboxyfluorescein diacetate; 79955-27-4; 5-CFDA; Cfda-5; 3',6'-diacetyloxy-3-oxospiro[2-benzofuran-1,9'-xanthene]-5-carboxylic acid; 3',6'-Diacetoxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-carboxylic acid; 5-Carboxy-di-O-acetylfluorescein; DTXSID00229971; CFDA; 5 CFDA; 5CFDA-5

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 本产品在运输和储存过程中需避光。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 100 mg/mL (~217.21 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.43 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (5.43 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (5.43 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。