6-CFDA N-succinimidyl ester (Carboxyfluorescein diacetate succinimidyl) is capable of spontaneously and irreversibly binding to free amines. Utilized in flow cytometry experiments for tracking cell division in both mammalian cells and bacteria. Used in combination with several cell permeabilizers as an effective, nondestructive fluorescencent stain to quickly detect bacterial cells.

Instructions
I. Solution preparation
1. Stock solution preparation: 6-CFDA N-succinimidyl ester is dissolved in anhydrous dimethyl sulfoxide (DMSO), usually prepared at a concentration of 1–10 mM.
2. Working solution preparation: Dilute the 6-CFDA solution to a concentration of 1–10 μM for use.
II. Operation steps
Cell labeling reaction
1. Cell treatment: Culture the cells to be labeled in a suitable growth environment. To increase the cell uptake of the dye, a cell permeabilizing agent (such as Triton X-100 or a surfactant) can be used.
2. Dye addition: Add the working solution to the culture medium and culture it with the cells. The cells are incubated in the labeling solution for 30 minutes to 1 hour at room temperature or 37°C.
3. Reaction termination: Wash the cells with a buffer containing the culture medium or PBS (phosphate-buffered saline) to remove unbound dye.
4. Detection and analysis:
a. Fluorescence microscopy: The labeled cells were observed using a fluorescence microscope with an excitation wavelength of 492 nm and an emission wavelength of 517 nm.
b. Flow cytometry: The fluorescence intensity of the cells was analyzed by flow cytometry to detect the labeling and division of the cells.
Cell division tracking:
1. In flow cytometry, 6-CFDA is often used in cell division experiments. After the labeled cells divide, the fluorescent signal is evenly distributed to the daughter cells, thereby tracking the cell division process.
Bacterial labeling: 6-CFDA can also be used for bacterial cell labeling. After the bacterial cells are permeabilized, the dye is able to enter the bacterial cells and label their internal molecules.