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Revision as of 15:48, 27 July 2009
CFSE is used to fluorescently label live cells in order to track them. 5,6-CFDA/SE is membrane permeable, but trapped in the cell when converted by intracellular esterases. CFSE is equally partitioned to daughter cells during division and can be used to measure cell proliferation.
T regulatory Assay
- 15 mL polypropylene tubes
- 5,6-CFDA/SE Invitrogen C1157 dissolved in DMSO at 5 mM and stored at -20°C.
Isolation of Mononuclear Cells
CFSE labelling of 7day culture cells
- Suspend PBMCs at 10x106 cells/mL in PBS alone.
- Ensure that cells are uniformly suspended when CFSE is added.
- If CFSE negative control is needed, remove cells now.
- Make '2X' concentration (10 μM) in PBS
- For example: add 5 mL PBS + 10 μL 5 mM CFSE
- Combine 1:1 PBMC cells + CFSE (e.g. 5 mL PBMCs + 5 mL CFSE) in 15mL tube and mix completely.
- Place in 37°C H2O bath x 10 min.
- Wash in 10 mL complete medium.
- Resuspend in medium at desired density
- Lyons AB and Parish CR. . pmid:8176234.
- Fuss IJ, Kanof ME, Smith PD, and Zola H. . pmid:19347849.
- Shreffler WG, Wanich N, Moloney M, Nowak-Wegrzyn A, and Sampson HA. . pmid:19130927.
- Who has experience with this protocol?
- Email Wayne Shreffler through OpenWetWare