| Size | Price | Stock | Qty |
|---|---|---|---|
| 100mg |
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| 250mg |
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| Other Sizes |
| Targets |
AFC is not a drug with a therapeutic target. It is a fluorescent compound used as a reporter to measure the activity of caspases (specifically caspase-3 and caspase-7), which cleave its parent compound, Ac-DEVD-AFC [1]
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| ln Vitro |
The fluorescent compound N-acetyl-asp-glu-val-asp-7-amino-4-(trifluoromethyl)coumarin (Ac-DEVD-AFC) was used to test caspase activators. The substrate penetrates the cell quickly and is effectively broken down at aspartic acid residues by certain caspases, resulting in the fluorescent molecule 7-amino-4-(trifluoromethyl)coumarin (AFC). The liberated 7-Amino-4-(trifluoromethyl)coumarin was isolated on HPLC and identified by fluorescence upon cell rupture. The intracellular caspase responsible for the emergence of intracellular 7-Amino-4-(trifluoromethyl)coumarin is identified [1] as a burst of the pancaspase benzyloxycarbonyl-val-ala-asp-fluoromethylketone. The synthesis of γ-glutamyl-7-amino-4-(trifluoromethyl)coumarin helped determine the fluorescence of γ-glutamyl transpeptidase by assisting the substrate reaction product 7-amino-4-(trifluoromethyl). With excitation and emission wavelengths of 400 and 490 nm, respectively, and a linear correlation with phosphors in the concentration range of 10-300 pmol/3 mL, coumarin responds linearly with phosphors at neutral pH. blend[2].
Role as a Caspase Activity Reporter: AFC is the fluorescent product generated when cellular caspases (e.g., caspase-3, caspase-7) cleave the synthetic fluorogenic substrate N-acetyl-asp-glu-val-asp-7-amino-4-trifluoromethyl coumarin (Ac-DEVD-AFC) at the aspartate residue. The amount of AFC produced is directly proportional to the level of intracellular caspase activity. This was validated by showing that AFC peak areas from HPLC were linear with caspase-3 concentration (R² = 0.972) and with incubation time for the first 3 hours (R² > 0.995) in a cell-free system. The appearance of AFC in cell-based assays was completely blocked by the pancaspase inhibitor zVAD-fmk, confirming that its generation is caspase-specific [1] . - Detection Method: AFC is separated from other cellular components and the parent substrate using reversed-phase HPLC. It is detected by its fluorescence, with an excitation wavelength of 400 nm and an emission wavelength of 505 nm. The area under the AFC peak in the chromatogram is quantified and used as a measure of caspase activity [1] . |
| Enzyme Assay |
In Vitro Caspase-3 Activity Assay: To validate the substrate, the activity of purified recombinant human caspase-3 was measured using Ac-DEVD-AFC. Reactions were carried out in a buffer containing sucrose, NaCl, Tris-HEPES (pH 7.5), dithiothreitol, EDTA, Ac-DEVD-AFC, and caspase-3, with or without the inhibitor zVAD-fmk. The mixture was incubated at 37°C for 15 minutes. The reaction was stopped, and samples were immediately analyzed by reversed-phase HPLC with fluorescence detection (excitation 400 nm, emission 505 nm). The resulting AFC peak areas were used to determine kinetic parameters and confirm the linearity of the assay with respect to enzyme concentration and time [1]
. - Determination of Kinetic Parameters: By measuring the initial rate of AFC production (V, derived from the HPLC peak area) at various concentrations of Ac-DEVD-AFC ([S]), a double-reciprocal (Lineweaver-Burk) plot of 1/V vs. 1/[S] was generated. From this plot, the Michaelis constant (Km) for caspase-3 with Ac-DEVD-AFC was calculated to be approximately 812 ± 478 μM [1] . |
| Cell Assay |
Intracellular Caspase Activity Measurement: To measure drug-induced apoptosis, cell suspensions (e.g., Jurkat, HL-60) at 10⁶ cells/mL were incubated with the fluorogenic substrate Ac-DEVD-AFC (68 μM) and various anticancer drugs (e.g., dactinomycin, doxorubicin, cisplatin). At specific time points, cells were lysed by sonication on ice and by passing through a fine-gauge needle. The lysate was centrifuged, and the supernatant was analyzed by HPLC with fluorescence detection to quantify the amount of free AFC produced. The AFC peak area, proportional to caspase activity, was plotted against incubation time to determine the kinetics of caspase activation ("caspase storm") [1]
. - Pulse-Chase Style Caspase Activity Assay: To measure caspase activity at a specific time point rather than cumulatively, cells were first incubated with a drug in the absence of Ac-DEVD-AFC. At the end of the desired incubation period, Ac-DEVD-AFC (68 μM) was added, and the incubation was continued for an additional 30 minutes. The amount of AFC generated during this 30-minute pulse was then quantified by HPLC, providing a snapshot of the caspase activity at that specific time [1] . |
| References | |
| Additional Infomation |
7-Amino-4-(trifluoromethyl)coumarin is a member of the 7-aminocoumarin family and is a fluorescent dye.
Role in Apoptosis Research: AFC is the fluorescent product of the widely used caspase substrate Ac-DEVD-AFC. Its quantification allows researchers to monitor the kinetics of caspase activation, a key executioner phase of apoptosis, in response to various stimuli such as anticancer drugs. The study describes the time-dependent "caspase storm" where AFC production, and thus caspase activity, rapidly increases after a lag phase and then decreases [1] . - Physicochemical Properties: AFC is a fluorescent compound that can be detected with high sensitivity using an excitation wavelength of 400 nm and an emission wavelength of 505 nm, allowing for its separation and quantification by HPLC [1] . |
| Molecular Formula |
C10H6F3NO2
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|---|---|
| Molecular Weight |
229.1554
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| Exact Mass |
229.035
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| CAS # |
53518-15-3
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| PubChem CID |
100641
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.5±0.1 g/cm3
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| Boiling Point |
314.6±42.0 °C at 760 mmHg
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| Melting Point |
221-222 °C(lit.)
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| Flash Point |
144.1±27.9 °C
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| Vapour Pressure |
0.0±0.7 mmHg at 25°C
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| Index of Refraction |
1.562
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| LogP |
1.64
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| Hydrogen Bond Donor Count |
1
|
| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
0
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| Heavy Atom Count |
16
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| Complexity |
337
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
JBNOVHJXQSHGRL-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C10H6F3NO2/c11-10(12,13)7-4-9(15)16-8-3-5(14)1-2-6(7)8/h1-4H,14H2
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| Chemical Name |
7-amino-4-(trifluoromethyl)chromen-2-one
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| HS Tariff Code |
2934.99.9001
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| Storage |
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. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~436.38 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (10.91 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 4.3638 mL | 21.8188 mL | 43.6376 mL | |
| 5 mM | 0.8728 mL | 4.3638 mL | 8.7275 mL | |
| 10 mM | 0.4364 mL | 2.1819 mL | 4.3638 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT06292806 | NOT YET RECRUITING | Drug: FSH-R | IVF Infertility, Female Ovulation Disorder |
Insemine Humen Reproduction Centre | 2024-03-01 | |
| NCT02430740 | RECRUITING | Drug: recFSH | Infertility | Cliniques universitaires Saint-Luc- Université Catholique de Louvain | 2016-01 | Phase 4 |
| NCT02739269 | COMPLETEDWITH RESULTS | Other: Serum AMH measurement Other: AFC measurement |
Infertility | The University of Hong Kong | 2016-04-07 | Not Applicable |
| NCT01783301 | COMPLETED | Drug: FSH | Infertility | Vietnam National University | 2011-10 | Phase 4 |
| NCT02354963 | COMPLETEDWITH RESULTS | Procedure: In vitro fragmentation of the ovarian tissue | Infertility | César Díaz García | 2014-11 | Not Applicable |
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