CFDA-SE is a fluorescent dye capable of cell membrane penetration. It reacts with free amine groups in the cytoskeleton proteins inside the cell to form a fluorescent protein complex. Once inside the cell, CFDA-SE localizes to the cell membrane, cytoplasm, and nucleus, with the most intense fluorescence staining present in the nucleus[1]. The CFDA-SE dye is consistently distributed to progeny during cell division and proliferation, with its dilution being proportional to the number of cell divisions. This dilution pattern can be monitored and quantified via flow cytometry using an excitation light of 488 nm, serving as a method for assessing cell proliferation[1].
体外研究
1. Preparation of CFDA-SE working solution
1.1 Preparation of CFDA-SE Reserve Solution. Reserve solutions should be prepared in dimethyl sulfoxide (DMSO, concentration 10 mM).Reserve solutions applied to unused solutions must be aliquoted and stored in a -20°C freezer. It is important to avoid repeated freeze/thaw cycles. It is important to avoid repeated freeze/thaw cycles. The solution can be stored for up to 6 months.
1.2 Preparation of CFDA-SE Working Solution
The stock solution should be diluted in serum-free cell culture, and it is recommended that it is used as it is prepared.
Please note that the final concentration of the working solution is dependent on the experimental system. It is therefore recommended that the final concentration conditions be optimised.
2. Cell Staining
2.1 Cell preparation
Suspension cells should be subjected to centrifugation at 1000 g for 3-5 minutes at 4°C. The upper layer should then be discarded, and the cells washed twice with PBS for 5 minutes on each occasion.
In order to proceed with the procedure, the cell culture medium must be discarded. Following this, trypsin should be added in order to dissociate the cells and create a single-cell suspension. The cells should then be subjected to centrifugation at 1000 g for 3-5 min at 4°C. The resultant pellet should then be discarded. The cells should be washed with PBS twice for 5 min each time.
2.2 The next step is to add 1 mL of CFDA-SE working solution and incubate for 30 minutes at room temperature.
2.3 The centrifuge should be set to 400 g and run for 3-4 minutes at 4°C.
2.4 The product should be washed twice with PBS, for a duration of five minutes on each occasion.
2.5 The cells should be resuspended in serum-free cell culture medium or PBS, and examined by fluorescence microscopy or flow cytometry.
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