6-FAM SE

6-FAM SE (6-carboxyfluorescein succinimidyl ester) is a fluorescent labeling reagent. 6-FAM SE is used for oligonucleotide labeling and DNA sequencing^[1]. In Vitro:1. Sample preparation^[1]:
1) Collect cells or biological samples to be stained. For cell samples, ensure that the cells are in the logarithmic growth phase. After collecting the cells, wash them twice with an appropriate buffer (such as PBS) to remove serum and other impurities in the culture medium.
2) Count the cells using a cell counter or hemocytometer and adjust the cell concentration to the appropriate range.
2. Preparation of dye stock solution:
1) Weigh an appropriate amount of 6-FAM SE powder and calculate the required mass based on its molecular weight and the required concentration. Generally speaking, the molecular weight of 6-FAM SE is about 467.4.
2) Dissolve 6-FAM SE powder in anhydrous DMSO to prepare a high concentration stock solution, usually 1-5 mM. For example, to prepare a 1 mM stock solution, dissolve 467.4 μg of 6-FAM SE in 1 mL of DMSO. Dispense the stock solution into sterile microcentrifuge tubes to avoid repeated freezing and thawing. It can be stored at -20°C or lower.
3. Preparation of working solution:
Before use, take out a tube of dye stock solution and thaw it at room temperature. Dilute the stock solution to the required working concentration, generally 0.5-5 μM, with cell culture medium or buffer preheated to 37°C. For example, take 10 μL of 1 mM stock solution and add it to 2 mL of culture medium to obtain 5 μM working solution.
4. Specific staining steps:
1) Mix the prepared cell sample with the dye working solution in an appropriate ratio. Generally, the volume ratio of cell suspension to working solution is 1:1 or 1:2. For example, take 1 mL of cell suspension and add 1-2 mL of dye working solution.
2) Mix gently to allow the cells to fully contact the dye and incubate in an incubator at 37°C and 5% CO₂ for 15-30 min. Gently shake or invert several times during this period to ensure uniform staining.
3) After incubation, wash the cells at least twice with pre-cooled buffer to remove unbound dye. After each wash, centrifuge at an appropriate speed (such as 300-500 g) for 5-10 min and discard the supernatant.
4) Finally, resuspend the stained cells in an appropriate amount of buffer or culture medium and use them for subsequent experimental analysis, such as flow cytometry detection, fluorescence microscopy observation, etc.