Fluorescent Dye

Preparation of 5(6)-CFDA Working Solution
1. Preparation of Stock Solution
Dissolve 1 mg of 5(6)-CFDA in 0.21 mL of DMSO to obtain a 10 mM 5(6)-CFDA solution.
Note: It is recommended to store the stock solution protected from light at -20°C or -80°C, avoiding repeated freeze-thaw cycles.
2. 5(6)-CFDA Working Solution
Dilute the stock solution with serum-free cell culture medium or PBS to prepare a 1-10 μM 5(6)-CFDA working solution.
Note: Adjust the concentration of the 5(6)-CFDA working solution according to experimental requirements.

Cell Staining
1. Cell Preparation:
• Suspension cells: Centrifuge at 1000 g for 3-5 minutes at 4°C, then discard the supernatant. Wash twice with PBS, 5 minutes each time.
• Adherent cells: Discard the cell culture medium, dissociate cells with trypsin to form a single-cell suspension. Centrifuge at 1000 g for 3-5 minutes at 4°C, then discard the supernatant. Wash twice with PBS, 5 minutes each time.
2. Add 1 mL of 5(6)-CFDA working solution and incubate at room temperature for 30 minutes.
3. Centrifuge at 400 g for 3-4 minutes at 4°C and discard the supernatant.
4. Wash twice with PBS, 5 minutes each time.
5. Resuspend the cells in serum-free cell culture medium or PBS, then analyze by fluorescence microscopy or flow cytometry.

Precautions
1. The stock solution should be stored protected from light at -20°C or -80°C, avoiding repeated freeze-thaw cycles.
2. Adjust the concentration of the 5(6)-CFDA working solution according to experimental requirements.
3. This product is intended for research use only and is not for drug, household, or other applications.
4. For your safety and health, wear a lab coat and disposable gloves during operation.

[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 (EPCs) are thought to be involved in aneurysm repair and remodeling. This study aimed to test this hypothesis and, if true, investigate how EPCs promote aneurysm repair in an elastase-induced rabbit carotid aneurysm model. Rabbits were randomly assigned to either the intracarotid EPC infusion group (ISCT group, n=5) or the intravenous EPC infusion group (IVT group, n=5). Autologous EPCs were double-labeled with Hoechst 33342 and 5,6-carboxyfluorescein diacetate succinimide, and then injected into the carotid artery (ISCT group) or the marginal auricular vein (IVT group), respectively. Three weeks later, the location, adhesion, and growth of labeled cells within the aneurysm were observed to detect signs of aneurysm repair. In the ISCT group, labeled EPCs were detected in the neointima of all five aneurysms; in the IVT group, labeled EPCs were detected in the neointima of three of the five aneurysms. However, no endothelial cell proliferation was observed in the neointima of either group of aneurysms. These results indicate that bone marrow-derived EPCs are involved in the repair process of aneurysms in this rabbit model. [1]

---

The ability of fluorescent dyes to stain live hyphae of two fungi—Neotyphodium lolii and Festuca arundinacea—was evaluated. Fluorescein-based fluorescent dyes: This study evaluated the staining effects of fluorescein diacetate (FDA), 5(6)-carboxyfluorescein diacetate (CFDA), 5-chloromethylfluorescein diacetate (CMFDA), and chitin-binding dye Calcofluor M2R on endophytic hyphae in sterile fungal cultures in vitro and in plants (including epidermal leaf sheath peels, nodes, ovaries, embryos, and meristems). CMFDA showed the highest staining intensity on fungal hyphae and exhibited excellent contrast in plants compared to plant cells. Compared to other dyes, CMFDA was least affected by photobleaching and continued to fluoresce for up to 2 hours after initial excitation. None of the fluorescent dyes could stain fungal hyphae in seeds. [2]

---

Endothelial progenitor cells (EPCs) are thought to be involved in aneurysm repair and remodeling. This study aimed to verify this hypothesis and explore how endothelial progenitor cells (EPCs) promote aneurysm repair in an elastase-induced rabbit carotid aneurysm model. Rabbits were randomly divided into two groups: the intracarotid EPC infusion group (ISCT group, n=5) and the intravenous EPC infusion group (IVT group, n=5). Autologous EPCs were double-labeled with Hoechst 33342 and 5,6-carboxyfluorescein diacetate succinimide and injected into the carotid artery (ISCT group) or the marginal auricular vein (IVT group), respectively. Three weeks later, the location, adhesion, and growth of labeled cells in the aneurysm were observed to detect signs of aneurysm repair. In the ISCT group, labeled EPCs were detected in the neointima of all 5 aneurysms; in the IVT group, labeled EPCs were detected in the neointima of 3 of the 5 aneurysms. However, no endothelial cell proliferation was observed in the neointima of either group of aneurysms. These results indicate that bone marrow-derived EPCs are involved in the aneurysm repair process in this rabbit model. [3]

2[C25H16O9]

920.778280000001

920.158

124387-19-5

5-CFDA;79955-27-4;6-CFDA;3348-03-6

44119974

White to yellow solid powder

7.606

2

18

10

68

1660

0

CC(OC1=CC=C2C(OC3=C(C=CC(OC(C)=O)=C3)C24C5=CC(C(O)=O)=CC=C5C(O4)=O)=C1)=O.CC(OC6=CC=C7C(OC8=C(C=CC(OC(C)=O)=C8)C79C%10=CC=C(C(O)=O)C=C%10C(O9)=O)=C6)=O

SWRGCNMDGRMQGB-UHFFFAOYSA-N

InChI=1S/2C25H16O9/c1-12(26)31-15-4-7-18-21(10-15)33-22-11-16(32-13(2)27)5-8-19(22)25(18)20-9-14(23(28)29)3-6-17(20)24(30)34-25;1-12(26)31-14-6-8-18-20(10-14)33-21-11-15(32-13(2)27)7-9-19(21)25(18)22-16(23(28)29)4-3-5-17(22)24(30)34-25/h2*3-11H,1-2H3,(H,28,29)

3',6'-diacetyloxy-1-oxospiro[2-benzofuran-3,9'-xanthene]-4-carboxylic acid;3',6'-diacetyloxy-1-oxospiro[2-benzofuran-3,9'-xanthene]-5-carboxylic acid

5(6)-Carboxyfluorescein diacetate; 124387-19-5; CFDA; 5-(6)-Carboxyfluorescein diacetate;CFDA; Spiro[isobenzofuran-1(3H),9'-[9H]xanthene]-ar-carboxylic acid, 3',6'-bis(acetyloxy)-3-oxo-; 3',6'-diacetyloxy-1-oxospiro[2-benzofuran-3,9'-xanthene]-4-carboxylic acid;3',6'-diacetyloxy-1-oxospiro[2-benzofuran-3,9'-xanthene]-5-carboxylic acid; 5-6-CFDA; DTXSID70657464;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~125 mg/mL (~271.51 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.52 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (4.52 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

 (Please use freshly prepared in vivo formulations for optimal results.)