5-CFDA (5-Carboxyfluorescein diacetate)(5-Carboxyfluorescein diacetate) is membrane-permeant. It can be loaded into cells via incubation and hydrolyzed by intracellular esterases to 5-carboxyfluorescein that is used for labeling human intervertebral disk cells in vitro for fluorescence microscopy.

Instructions
I. Solution preparation
1. Stock solution: Dissolve 5-CFDA in anhydrous DMSO to prepare a stock solution with a concentration of 1–10 mM.
Note: Keep away from light during operation. Store the stock solution at -20°C after aliquoting. Avoid repeated freezing and thawing to ensure stability.
2. Working solution: Dilute the stock solution to the final use concentration (1–10 µM). Phenol red-free cell culture medium or PBS buffer can be used for dilution.
II. Operation steps
1. Cell preparation:
Collect the adherent or suspension cells to be treated according to the experimental requirements and adjust to the appropriate cell density; rinse the cells 1–2 times with PBS or phenol red-free culture medium to remove substances that may interfere with the experiment.
2. Staining reaction:
Add the prepared 5-CFDA working solution to the cell suspension or culture medium to ensure that the dye is in full contact with the cells: usually at a concentration of 1–10 µM, incubate at 37°C in the dark for 15–30 minutes.
3. Washing steps: Wash the cells 2–3 times with PBS or fresh culture medium to remove excess dye that is not absorbed by the cells.
4. Detection and result analysis
1) Fluorescence signal detection: Detection is performed using a fluorescence microscope, flow cytometer, or fluorescence plate reader. Recommended parameters are:
Excitation wavelength: 490–495 nm
Emission wavelength: 515–525 nm
2) Data processing:
Compare the fluorescence intensity of the experimental group and the control group to evaluate cell activity, track cells, or analyze other biological dynamic changes.