6-CFDA

Biological Activity

6-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 6-CFDA working solution

1.Preparation of the stock solution
Dissolve 1mg 6-CFDA in 0.2172 mL DMSO to obtain 10 mM of 6-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 6-CFDA working solution
Dilute the stock solution in serum-free cell culture medium or PBS to obtain 1-10 μM of 6-CFDA working solution.
Note: Please adjust the concentration of 6-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 6-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 6-CFDA working solution according to the actual situation.

3. For your safety and health, please wear a lab coat and disposable gloves to operate.

Chemical Information

Molecular Weight	460.39
Formula	C25H16O9
CAS Number	3348-03-6
Solubility (25°C)	DMSO 90 mg/mL
Storage	-20°C, protect from light

Conversion of different model animals based on BSA (PMID: 27057123)

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 K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.

References

[1] Iigo Terrn, et al. Methods Enzymol. CFSE dilution to study human T and NK cell proliferation in vitro

[2] Mengting Jiang, et al. J Vis Exp. Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

[3] Chih-Ju Lin, et al. Biomed Opt Express. Direct visualization of functional heterogeneity in hepatobiliary metabolism using 6-CFDA as model compound

[4] A Bruce Lyons, et al. Curr Protoc Cytom. Flow cytometric analysis of cell division by dilution of CFSE and related dyes

[5] Ben J C Quah, et al. Nat Protoc. Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester