CFSE Cat. No. 13-0850-U500
Note: CFSE is provided as 500 ug of lyophilized powder. CFSE may be reconstituted to a stock concentration of 5 mM with 180 µL of anhydrous DMSO. It is recommended to aliquot smaller volumes and store at 20°C with dessicant and protected from light. Avoid repeated freeze-thaw cycles.
Other Materials Required
- 1X PBS ∙ RPMI complete medium (RPMI with 10% FBS, 1% pen/strep, 50 µM 2ME)
- Prepare cells as a single cell suspension.
- Wash cells twice with 1-2 mL 1X PBS to remove serum. Spin at 300-400 x g for 5 minutes at room temperature and decant supernatant.
- Resuspend cells at 1-10 x 10^6 /mL in room temperature 1X PBS.
- Prepare a working solution of CFSE at 2X the desired final concentration in room temperature 1x PBS (ie: for a final concentration of 5 µM CFSE, prepare a 2X working solution by adding 2 µL of 5mM stock to 1 mL 1X PBS). Note: CFSE can be used to label cells at concentrations ranging from 0.5-20 µM, depending on the cell type and assay. It is recommended that the investigator determine the optimal concentration of CFSE for the assay of interest. Over-labeling of cells with high concentrations of CFSE can obstruct normal cell functions and interfere with compensation in multicolor experiments.
- Add an equal volume of 2X CFSE working solution to cell preparation.
- Mix immediately and incubate in the dark for 10-20 minutes at room temperature.
- Quench the labeling reaction by adding 5 volumes of cold complete media and incubate on ice for 5 minutes, protected from light.
- Centrifuge the cells at 300-400 x g for 5 minutes at room temperature and decant supernatant. Wash cells 2 times with 1-2 mL complete media.
- CFSE labeled cells are ready for assay.