CFSE

Preparation of CFDA-SE working solution
1.1 Preparation of the stock solution
Dissolve 1 mg of CFDA-SE in 0.1794 mL of DMSO to obtain 10 mM of CFSE.
Note: It is recommended to store the stock solution at -20 ℃ or -80 ℃ away from light and avoid repetitive freeze-thaw cycles.
1.2 Preparation of CFDA-SE working solution
Dilute the stock solution in serum-free cell culture medium or PBS to obtain 5-10 μM of CFDA-SE working solution.
Note: Please adjust the concentration of CFDA-SE working solution according to the actual situation.
Cell staining
2.1 For suspension cells: Centrifuge at 1000 g at 4℃ for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time.
For adherent cells: Discard the cell culture medium, and add trypsin to dissociate cells to make a single-cell suspension. Centrifuge at 1000 g at 4℃ for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time.
2.2 Add 1 mL of CFDA-SE working solution, and then incubate at room temperature for 30 minutes.
2.3 Centrifuge at 400 g at 4℃ for 3-4 minutes and then discard the supernatant.
2.4 Wash twice with PBS, 5 minutes each time.
2.5 Resuspend cells with serum-free cell culture medium or PBS, and then detect by fluorescence microscope or flow cytometer.

The 5 mM CFDA-SE stock in DMSO is diluted to different concentrations(2 µM, 3 µM, 4 µM, 5 µM, 10 µM and 20 µM) in PBS with a total volume of 1 ml. After each cell line is harvested and washed three times with PBS, 1×10⁹ cells are added to equal volume of CFDA-SE with different concentrations and incubated at 37°C for 5, 6, 7, 8, 10 and 15 min with agitation. The labeling reaction is stopped for 1 min by adding an equal volume of heat inactivated fetal bovine serum. The CFDA-SE labeled cells are washed twice with PBS and recounted, and the cell concentration is adjusted to 6×104cells/ml in IMDM containing 10% FCS.

In vivo labeling of cells with CFSE is feasible with virtually no toxic effect on the labeled or adjacent cells. It has advantages in relatively long-term migration studies as demonstrated by the recovery of T-cells in the peripheral lymphoid organs^[2].