6-CFDA, recognized as an aliphatic luciferin-like entity, permeates cells freely, converting into carboxyl fluorescein (CF) via intracellular non-specific lipase activity. This transformation endows CF with an extra negative charge, enhancing its cellular retention relative to other fluorescein dyes[1][2][3].
体外研究
1. Preparation of 6-CFDA Working Solution
1.1 The first step is to prepare a 6-CFDA Reserve Staining Solution. The stock solution should be prepared in dimethyl sulfoxide (DMSO, concentration 10 mM. Any unused solution for stock solution application must be aliquoted and stored at a temperature of at least -20°C. It is important to avoid repeated freeze/thaw cycles. The solution can be stored for up to 6 months.)
1.2. Preparation of working solution: The stock solution should be diluted with pre-warmed serum-free cell culture medium or PBS to create a 1-10 μM working solution of 6-CFDA. It is recommended that the working solution be 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:
The process for collecting suspension cells involves centrifugation, followed by two washes with PBS, each lasting five minutes.
Adherent cells: The medium should then be discarded and trypsin added in order to digest the cells. Following centrifugation to remove the upper layer, PBS should be added and the sample washed twice, with a duration of five minutes per wash.
2.2. Next, add 1 mL of 6-CFDA working solution and incubate for 15 minutes at room temperature.
2.3. Centrifuge at 400 g for 3-4 minutes at 4°C. Please dispose of the item immediately.
2.4. Please add PBS and proceed to wash the cells twice, for a duration of five minutes on each occasion.
2.5. The cells should be resuspended in 1 mL of serum-free medium or PBS, and examined under a fluorescence microscope or flow cytometer.
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