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.

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].

• Cell lines

  human erythroleukaemic cell line K562, mouse lymphoma cell line YAC-1, human mammary cancer cell line MCF-7 and human melanoma cell line A375

• Concentrations

  2 µM, 3 µM, 4 µM, 5 µM, 10 µM and 20 µM

• Incubation Time

  5, 6, 7, 8, 10 and 15 min

• Method

  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.

• Animal Models

  C57Bl/6 mice

• Dosages

  10 μM

• Administration

  injected into thymic lobe