Fluorescent Dye

Preparation of 5-CFDA Working Solution
1. Stock Solution Preparation
• Dissolve 5-CFDA in DMSO to prepare a 10 mM stock solution.
Example: Dissolve 1 mg 5-CFDA in 0.2172 mL DMSO.
Note: Aliquot and store the stock solution at -20°C or -80°C, protected from light.

2. Working Solution Preparation
• Dilute the stock solution with pre-warmed serum-free cell culture medium or PBS to prepare a 1-10 μM 5-CFDA working solution.
Note: Adjust the working concentration as needed and prepare fresh before use.
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Cell Staining Protocol
1. Cell Preparation
• Suspension cells: Centrifuge to pellet cells, wash twice with PBS (5 min each).
• Adherent cells: Remove medium, detach cells with trypsin. After centrifugation, discard supernatant and wash twice with PBS (5 min each).
2. Staining
• Add 1 mL of 5-CFDA working solution and incubate at room temperature for 15 min.
3. Washing
• Centrifuge at 400 × g, 4°C for 3-4 min, discard supernatant.
• Wash cells twice with PBS (5 min each).
4. Resuspension & Detection
• Resuspend cells in 1 mL serum-free medium or PBS.
• Analyze using fluorescence microscopy or flow cytometry.
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Precautions
1. Storage: Aliquot 5-CFDA stock solution and store at -20°C (1 month) or -80°C (6 months). Avoid repeated freeze-thaw cycles. Protect from light.
2. Concentration: Optimize the working solution concentration based on experimental conditions.
3. Use: For research purposes only. Not for clinical diagnosis, therapy, food, or drug use.
4. Safety: Wear a lab coat and disposable gloves during handling.

[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. Labeled EPCs were detected in the neointima of all 5 aneurysms in the ISCT group and in 3 of the 5 aneurysms in the IVT group, but no endothelial cell proliferation was observed in the neointima of the aneurysms in either group. These results indicate that bone marrow-derived EPCs are involved in the aneurysm repair process in this rabbit model. [1]
We evaluated the ability of fluorescent dyes to stain the live hyphae of two fungi—Neotyphodium lolii and Festuca arundinacea—symbiotic endophytes of perennial ryegrass (Lolium perenne) and tall fescue (Festuca arundinacea), respectively. Fluorescein-based fluorescent dyes: This study evaluated the staining effects of fluorescein diacetate (FDA), 5(6)-carboxyfluorescein diacetate (CFDA), 5-chloromethylfluorescein diacetate (CMFDA), and the 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 with other dyes, CMFDA is least affected by photobleaching and continues to fluoresce for up to 2 hours after initial excitation. None of the fluorescent dyes could stain the fungal hyphae in the seeds. [2]

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Endothelial progenitor cells (EPCs) are thought to be involved in the repair and remodeling of aneurysms. 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 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 aneurysm repair process 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

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

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 : ≥ 100 mg/mL (~217.21 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.43 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 25.0 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.43 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 25.0 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.43 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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