Shreffler:CFSE Labeling

Overview
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.

Materials

 * 15 mL polypropylene tubes
 * PBS
 * 5,6-CFDA/SE Invitrogen C1157 dissolved in DMSO at 5 mM and stored at -20°C.

Procedure

 * 1) Suspend PBMCs at 10x106 cells/mL in PBS alone.
 * 2) Ensure that cells are uniformly suspended when CFSE is added.
 * 3) If CFSE negative control is needed, remove cells now.
 * 4) Make '2X' concentration (10 μM) in PBS
 * 5) *For example: add 5 mL PBS + 10 μL 5 mM CFSE
 * 6) Combine 1:1 PBMC cells + CFSE (e.g. 5 mL PBMCs + 5 mL CFSE) in 15mL tube and mix completely.
 * 7) Place in 37°C H2O bath x 10 min.
 * 8) Wash in 10 mL complete medium.
 * 9) Resuspend in medium at desired density

Discussion
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Contact

 * Who has experience with this protocol?
 * Email Wayne Shreffler through OpenWetWare