5-CFDA is a common aliphatic luciferin-line organism. CFDA conducts free diffusion into cells, and then it is hydrolyzed into carboxyl fluorescein (CF) by intracellular non-specific lipase.
Preparation of 5-CFDA working solution
1.Preparation of the stock solution
Dissolve 1mg 5-CFDA in 0.2172 mL DMSO to obtain 10 mM of 5-CFDA.
Note: It is recommended to store the stock solution at -20°C -80°C away from light and avoid repetitive freeze-thaw cycles.
2.Preparation of 5-CFDA working solution
Dilute the stock solution in serum-free cell culture medium or PBS to obtain 1-10 μM of 5-CFDA working solution.
Note: Please adjust the concentration of 5-CFDA working solution according to the actual situation.
Cell staining
1.Cell preparation:
For suspension cells: Centrifuge at 1000 g at 4°C for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time.
For adherent cells: Discard the cell culture medium, and add trypsin to dissociate cells to make a single-cell suspension. Centrifuge at 1000 g at 4°C for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time.
2.Add 1 mL of 5-CFDA working solution, and then incubate at room temperature for 30 minutes.
3.Centrifuge at 400 g at 4°C for 3-4 minutes and then discard the supernatant.
4.Wash twice with PBS, 5 minutes each time.
5.Resuspend cells with serum-free cell culture medium or PBS, and then detect by fluorescence microscope or flow cytometer.
Precautions
1. It is recommended to store the stock solution at -20℃ or -80℃ away from light and avoid repetitive freeze-thaw cycles.
2. Please adjust the concentration of 5-CFDA working solution according to the actual situation.
3. For your safety and health, please wear a lab coat and disposable gloves to operate.
| Molecular Weight | 460.39 |
| Formula | C25H16O9 |
| CAS Number | 79955-27-4 |
| Solubility (25°C) | DMSO ≥ 90 mg/mL |
| Storage | -20°C, protect from light |
| Species | Mouse | Rat | Rabbit | Guinea pig | Hamster | Dog |
| Weight (kg) | 0.02 | 0.15 | 1.8 | 0.4 | 0.08 | 10 |
| Body Surface Area (m2) | 0.007 | 0.025 | 0.15 | 0.05 | 0.02 | 0.5 |
| Km factor | 3 | 6 | 12 | 8 | 5 | 20 |
| Animal A (mg/kg) = Animal B (mg/kg) multiplied by | Animal B Km |
| Animal A Km |
For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the Km factor for a mouse and then divide by the Km factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.
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