5(6)-CFDA (5-(6)-Carboxyfluorescein diacetate) is a cell-permeant esterase substrate that can serve as a viability probe that measures both enzymatic activities, which is required to activate its fluorescence, and cell-membrane integrity, which is required for intracellular retention of their fluorescent product.

Instructions
I. Reagent preparation
Preparation of stock solution and working solution: Prepare stock solution by dissolving with an appropriate solvent (e.g., dimethyl sulfoxide (DMSO)); the stock solution concentration is usually 1–10 mM, and then dilute it to the working concentration (usually 1–5 μM) according to the experimental requirements.
II. Operation steps
Cell staining process
1. Cell incubation:
1) Inoculate the cells to be tested into a culture dish or multi-well plate.
2) Add 5(6)-CFDA solution to the cells, ensuring that the concentration is the working concentration (usually 1–5 μM), and incubate at 37°C for 30 minutes to 1 hour. The incubation time can be adjusted according to the cell type and experimental requirements.
2. Washing: After incubation, wash the cells with appropriate culture medium or PBS to remove unhydrolyzed dye.
3. Cell imaging: After washing, the cells can be analyzed by fluorescence microscopy or other imaging equipment. 5(6)-CFDA will emit strong green fluorescence under 492 nm excitation, which is convenient for observing the stained cells.
4. Flow cytometry: Flow cytometry analysis is performed on the stained cells, and the fluorescence intensity can be used to evaluate the activity, proliferation or other related characteristics of the cells.
5. e. v Data analysis:
1) According to the change in fluorescence intensity, the activity, proliferation and other processes of the cells can be evaluated.
2) The data can be further analyzed by comparing the standard curve or the control experimental group with the experimental group.
6. Application:
1) Cell survival and proliferation detection: 5(6)-CFDA is often used in cell survival and proliferation detection. After the cells hydrolyze the dye, the fluorescence intensity is proportional to the number of cells.
2) Cell uptake study: It can be used to study the cell uptake of the dye and its distribution in the cell.
3) Long-term labeling: Since the dye is hydrolyzed by the cells, it can be used to label cells for a long time, which has application value in proliferation experiments and long-term culture studies.
Notes:
1. Cell permeability: 5(6)-CFDA is cell permeable, but its hydrolysis into fluorescent products depends on the activity of intracellular esterases. Therefore, only living cells will show fluorescence.
2. Solvent usage: Make sure that the solvent used to dissolve the dye (such as DMSO) does not affect cell activity.
3. Photosensitivity: 5(6)-CFDA is light-sensitive, so strong light exposure should be avoided during operation and storage to prevent fluorescence bleaching.