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Principles and Precautions for CFDA‑SE Cell Proliferation Tracing Using Fluorescent Probes
CFDA-SE It can be used for cell tracking as well as for assessing cell proliferation. Cells labeled with CFDA‑SE are suitable for both in vitro and in vivo proliferation studies and do not stain neighboring cells. CFDA‑SE is most commonly employed to measure lymphocyte proliferation, but it can also be applied to assess the proliferation of fibroblasts, natural killer cells, hematopoietic progenitor cells, and other cell types.
Detection principle
CFDA SE can penetrate intact cell membranes and, once inside the cell, is hydrolyzed by intracellular esterases into CFSE. CFSE undergoes random, irreversible covalent binding to lysine residues or other amino groups on intracellular proteins, thereby labeling these proteins; cells labeled with CFDA‑SE exhibit green fluorescence. In dividing cells labeled with CFDA‑SE, the fluorescence intensity decreases by half with each division: after one division, the fluorescence is reduced to 1/2; after two divisions, it is further reduced to 1/4; and so on, with the fluorescence intensity progressively diminishing according to the sequence 1/2, 1/4, 1/8, 1/16, and so forth.
Instructions for Use
1. Preparation of the preservation solution (5 mM)
Prepare as needed: pipette 360 μL of DMSO to dissolve 1 mg of CFSE, then sonicate to ensure complete dissolution, yielding a 5 mM CFSE stock solution. Aliquot this solution into dark‑protected, sterile cryovials at 40 μL per vial; it can be stored at −20°C for up to 2 months.
2. Preparation of the working solution
Take 1 μL of the 5 mM CFSE stock solution and dilute it in 1 mL of PBS containing 10% FCS.
3. Dyeing
(1) Resuspend the cells. Resuspend the cells in PBS containing 5% FCS, at a cell concentration typically of 1 × 10^6 cells/mL for in vitro experiments or 1 × 10^7 cells/mL for transfection experiments.
(2) Staining. Mix 1 mL of DFSE working solution with 1 mL of cell suspension and incubate at 37°C for 5–10 minutes, gently mixing twice during this period.
(3) Terminate staining. Wash three times with complete culture medium and centrifuge. Typically, after the second wash, incubate for an additional 5 minutes before performing the third wash. Resuspend gently in ice‑cold RPMI‑1640 medium containing 10% fetal bovine serum and L‑glutamine (catalog no.: AC01L064), mixing lightly for 1 minute (by gently tapping the tube; avoid pipetting or vigorous mixing), then centrifuge at 1,500 × g for 5 minutes.
(4) Flow cytometry was performed in the FL1 channel.
Precautions
✔ All reagents must be thoroughly thawed and mixed before use; during mixing, minimize bubble formation. The CFDA SE solvent may solidify at lower temperatures—incubate in a 20–25°C water bath for a few minutes until completely dissolved before use.
✔ The reagent contains a fluorescent dye; during storage or labeling, handle it away from light as much as possible to prevent fluorescence quenching.
✔ Lipase activity varies among different cell types, resulting in differences in staining intensity. The optimal working concentration can be determined by optimizing conditions based on the cell type and culture environment.
✔ If cell fixation is required, use an aldehyde-based fixative such as 3.7% paraformaldehyde at room temperature for 15 minutes; thereafter, if additional procedures such as antibody labeling are needed, permeabilize the cells with ice-cold acetone for 10 minutes.
✔ For your safety and health, please wear a lab coat and disposable gloves while performing this procedure.
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