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.
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.
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.
|Solubility (25°C)||DMSO 90 mg/mL|
|Storage||-20°C, protect from light|
|Body Surface Area (m2)||0.007||0.025||0.15||0.05||0.02||0.5|
|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.
 Iigo Terrn, et al. Methods Enzymol. CFSE dilution to study human T and NK cell proliferation in vitro
 Mengting Jiang, et al. J Vis Exp. Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis
 Chih-Ju Lin, et al. Biomed Opt Express. Direct visualization of functional heterogeneity in hepatobiliary metabolism using 6-CFDA as model compound
 A Bruce Lyons, et al. Curr Protoc Cytom. Flow cytometric analysis of cell division by dilution of CFSE and related dyes
 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
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