CFDA-SE

1. Preparation of staining solution

(1) Preparation of stock solution: dissolve CFDA-SE in DMSO to prepare a stock solution, typically at a concentration of 10mM. Stock solution are recommended to be aliquoted and stored at -20°C or -80°C protected from light. Avoid repeated freeze-thaw cycles.

(2) Preparation of working solution: dilute the stock solution with serum-free medium or PBS to prepare a working solution at 1-10μM. Since CFDA-SE hydrolyzes rapidly in aqueous solutions, prepare the working solution immediately before use. Proteins present in serum and culture media compete with CFDA-SE for binding, leading to a substantial decrease in labeling efficiency. The preparation of working solutions and the labeling process must be strictly performed under serum-free conditions.

2. Staining of Suspension Cells

(1) Cell collection: centrifuge at 1000×g for 3-5min at 4℃, discard the supernatant, and wash the cells twice with pre-warmed (37℃) serum-free PBS, 5min each time.

(2) Cell density adjustment: resuspend the cells in serum-free PBS and adjust the density to 1×10⁶-1×10⁷cells/mL.

(3) Staining: add CFDA-SE working solution (ensure the final concentration reaches the working concentration) and mix quickly and gently.

(4) Incubation: incubate the cell suspension at 37℃ in the dark for 10-30min. The optimal incubation time varies among different cell types and should be determined empirically based on specific experimental requirements.

(5) Washing and resuspension: after incubation, centrifuge at 1000×g for 5min, discard the supernatant, and wash the cells twice with PBS, 5min each time, to remove residual free dye.

3. Staining of Adherent Cells (Suspension Labeling Method)

(1) Cell collection: remove the culture medium, wash the cells twice with PBS, and detach the cells using trypsin. After digestion, centrifuge at 1000×g for 3-5min.

(2) Follow steps (2) through (5) under "Staining of Suspension Cells".

4. Staining of Adherent Cells (In Situ Labeling Method)

(1) Plating: culture cells on plates until they reach approximately 60%-80% confluence.

(2) Washing: remove the old medium and wash the cells twice with pre-warmed serum-free PBS.

(3) Incubation: add CFDA-SE working solution to just cover the cells, according to experimental requirements. Incubate at 37℃ in the dark for 10-30min. The optimal incubation time varies among different cell types and should be determined empirically.

(4) Washing: aspirate the staining working solution and wash the cells twice with PBS.

5. Detection

Depending on the experimental purpose, detach the cells if needed, and observe under a fluorescence microscope or by flow cytometry at Ex/Em 488/518nm.

Notes:

(1) Intracellular esterase activity varies among different cell types, which may result in differential staining efficiency.

(2) Fluorescent dyes are susceptible to photobleaching. Protect from light throughout the staining procedure.