5-(and 6)-carboxyfluorescein diacetate, succinimidyl ester (5,6 CFDA,SE; CFSE), is a fluorogenic reagent which is frequently used in cell labeling and cell proliferation procedures.
Non-fluorescent and moderately hydrophobic in its di-esterified form, CFDA-SE is colorless, non-fluorescent, and membrane permeable. After diffusing through the cell membrane’s lipid bi-layers into living animal cells, the acetate groups on CFDA-SE are cleaved by intracellular esterase enzymes to yield the amine-reactive and highly fluorescent product, carboxyfluorescein succinimidyl ester.
CFDA-SE easily penetrates the lipid bi-layers of living cells and is quickly converted to CFSE, the green fluorescent membrane-impermeant form, upon cleavage of the acetate groups by intracellular esterases. This highly fluorescent label is retained on and within the cells, while any excess CFSE probe is easily washed away in subsequent wash steps.
CFSE dye levels in non-dividing cells remain relatively stable. In dividing cells, the dye is divided equally at each generation, allowing quantitative proliferation studies with the use of a flow cytometer (FL1).
The high retention efficiency of the dye makes it useful for cell population identification. Since the cleaved form of CFSE is membrane impermeant, this cellular probe is useful for cell labeling and tracking applications. The reduction in CFSE fluorescence per cellular generation may be tracked for cell proliferation studies.
1. Reconstitute the vial of CFSE with 200 µL DMSO to create a 2500X stock concentrate at 0.25 mg/mL. Mix by swirling or tilting the vial, allowing the DMSO to travel around the base of the amber vial until completely dissolved. At room temperature, the reagent should be dissolved within a few minutes.
2. If storing the stock concentrate for future use, prepare small aliquots (20 µL) to avoid freeze-thaw cycles. The stock concentrate will be stable for 6 months when protected from light and stored at or below -20°C.
3. Create the 10X working solution by diluting the 2500X stock solution 1:250 in sterile PBS; e.g., add 4 µL stock to 996 µL PBS. Store the working solution on ice up to 2 hours protected from light. Do not use media to dilute the CFSE as it will quench the fluorescent signal.
4. Prepare cells at 1-2 x 107 in 1.8 mL sterile PBS.
5. Create 1 control tube of unstained cells at 1-2 x 107 in 2 mL sterile PBS. These cells will be used to compensate the flow cytometer to ensure that stained cells shift along the FL1 axis.
6. Stain cells at a final concentration of 0.1 µg/mL (0.18 µM) of CFSE in the cell culture. Add the 10X working solution to the cells at a dilution of 1:10. For example, add 200 µL 10X CFSE working solution into 1.8 mL cell suspension. Mix by inverting or vortexing the vial. The optimal concentration of CVSE may vary among cell types. The concentration of CFSE and the incubation time should be adjusted for cell line to adequately stain them. Excessive staining may cause problems when compensating the instrument. Do not add CFSE to the control tube.
7. Incubate 15 minutes at room temperature.
8. Add 1 mL cell culture media to stop the reaction.
9. Incubate 5 minutes.
10. Wash the cells once or twice by centrifugation and discard the supernatant.
11. Resuspend in cell culture media such that each tube contains the desired level of target cells, or resuspend in PBS and fix cells with formaldehyde.
12. Analyze cells, or incubate at 37°C up to 1 hour until ready for additional staining or further experimentation.
13. Analyze with a flow cytometer equipped with a 15 mW 488 nm argon laser: excitation at 492 nm; emission at 520-540 nm in FL1. Stained cells appear green.
• Molecular weight: 557.47
• Excitation/Emission: 492 nm / 520-540 nm
• Method of Analysis: Flow cytometer, Fluorescence Microscope
• Storage: -20°C
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