CFSE Green Fluorescent Stain - for cell labelling and proliferation studies

1 Citation

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, the dye easily penetrates lipid bi-layers of living cells and is quickly converted to the amine-reactive, green fluorescent form (CFSE) upon cleavage of the acetate groups by intracellular esterases. This highly fluorescent form is membrane-impermeant and is retained on and within the cells, while any excess probe is easily washed away in subsequent wash steps. Because CFSE forms a strong bond inside the cell, it is retained within the cell indefinitely and is inherited by daughter cells. It will not be incorporated into adjacent cells.

Figure 1. Staining of Healthy Jurkat Cells Healthy Jurkat cells were stained with CFSE or left unstained and then analyzed by flow cytometry using an Accuri C6 flow cytometer equipped with a FL1 99% attenuation filter. Unstained Jurkat cells (A), stained Jurkat cells (B), and the corresponding overlay (C) are shown.
Figure 2. Identification of CFSE-stained Cells. K562 target cells were stained green with CFSE and then subjected to effector cells. Green stained target cells are easily identifiable when analyzed by FL1 vs. SSC on a flow cytometer. Target cells stained green with CFSE move to the right along the X-axis (FL1) of the plot (right) compared with unstained effector cells. This plot becomes particularly important when gating on target cells that are the same size as effector cells.
Figure 3. K562 target cells were stained with CFSE to distinguish them from effector cells in FL1 (Fig 2). The target cells were stained with 7-AAD (cat 6163) to identify membrane-compromised necrotic target cells (red in FL3), and with SR-FLICA® (cat 917) to identify caspase-positive apoptotic target cells (orangy-red in FL2). When used together with Total Cytotoxicity Assay kit (cat 972), these reagents identified four populations of cells: live; early apoptotic; late apoptotic; and necrotic.
Figure 1. Staining of Healthy Jurkat Cells Healthy Jurkat cells were stained with CFSE or left unstained and then analyzed by flow cytometry using an Accuri C6 flow cytometer equipped with a FL1 99% attenuation filter. Unstained Jurkat cells (A), stained Jurkat cells (B), and the corresponding overlay (C) are shown.
Figure 1. Staining of Healthy Jurkat Cells Healthy Jurkat cells were stained with CFSE or left unstained and then analyzed by flow cytometry using an Accuri C6 flow cytometer equipped with a FL1 99% attenuation filter. Unstained Jurkat cells (A), stained Jurkat cells (B), and the corresponding overlay (C) are shown.
Figure 2. Identification of CFSE-stained Cells. K562 target cells were stained green with CFSE and then subjected to effector cells. Green stained target cells are easily identifiable when analyzed by FL1 vs. SSC on a flow cytometer. Target cells stained green with CFSE move to the right along the X-axis (FL1) of the plot (right) compared with unstained effector cells. This plot becomes particularly important when gating on target cells that are the same size as effector cells.
Figure 3. K562 target cells were stained with CFSE to distinguish them from effector cells in FL1 (Fig 2). The target cells were stained with 7-AAD (cat 6163) to identify membrane-compromised necrotic target cells (red in FL3), and with SR-FLICA® (cat 917) to identify caspase-positive apoptotic target cells (orangy-red in FL2). When used together with Total Cytotoxicity Assay kit (cat 972), these reagents identified four populations of cells: live; early apoptotic; late apoptotic; and necrotic.

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Data Sheet Safety Data Sheet

SKU: 6162

Size: 50 µg
1-2 business days
Price:
$120.75

CFSE is a green fluorogenic reagent that binds to intracellular molecules. It is often used for cell proliferation and cytotoxicity studies. CFSE diffuses into the cell and covalently binds to primary amino groups present on intracellular molecules. Intracellular esterases quickly cleave the acetate groups from the dye thus converting it to the fluorescent form. Any unbound reagent diffuses back out of the cell. Because CFSE forms a strong bond inside the cell, it is retained within the cell indefinitely and is inherited by daughter cells. It will not be incorporated into adjacent cells.

CFSE is supplied as a concentrated lyophilized powder at 0.05 mg. Reconstitute it with 200 µL DMSO to yield a stock concentrate at 2500X (0.25 mg/mL). Dilute it 1:250 in PBS to form the 10X working solution, and then add it to cells at 1:10 (a final concentration of 0.1 µg/mL). Analyze with a fluorescence microscope or flow cytometer with a 488 nm blue argon excitation laser. CFSE exhibits green fluorescence in the FL1 region: excitation at 492 nm and emission at 520-540 nm (Figures 1-3).

As CFSE is detected in the green range, it is optimal for use in dual- staining studies with other fluorescent reagents, such as PI (catalog #638), 7-AAD (catalog #6163), and SR-FLICA® reagents with minimal spectral overlap. It can be used with our SR-FLICA® poly caspases inhibitor reagent (catalog #917) to identify apoptotic cells in the cell sample (Figure 3).

492 nm / 520-540 nm
Flow cytometry, Fluorescence microscope
-20°C
Domestic: Overnight Delivery; International: Priority Shipping
United States
    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 FL.
    14. Stained cells appear green.

Product Specific References

PMID Publication
38773691Cheng, O.J., et al. 2024. Mucosal-associated invariant T cells modulate innate immune cells and inhibit colon cancer growth. Scandinavian journal of immunology, e13391.

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