Shreffler:CFSE Labeling: Difference between revisions
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*15 mL polypropylene tubes | *15 mL polypropylene tubes | ||
*PBS | *[[PBS]] | ||
*5,6-CFDA/SE [http://products.invitrogen.com:80/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&entryPoint=adirect&productID=C1157&messageType=catProductDetail Invitrogen C1157] dissolved in DMSO at 5 mM and stored at -20°C. | *5,6-CFDA/SE [http://products.invitrogen.com:80/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&entryPoint=adirect&productID=C1157&messageType=catProductDetail Invitrogen C1157] dissolved in DMSO at 5 mM and stored at -20°C. | ||
Revision as of 18:58, 19 May 2009
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
Suspend PBMCs at 10x106 cells/mL in PBS alone.
Cells should be in PBS for CFSE labeling.
If CFSE negative control is needed, remove cells now.
Make '2X' concentration (10 μM) in PBS
- Add 5 mL PBS + 10 μL 5 mM CFSE (for example)
Combine 1:1 PBMC cells + CFSE (e.g. 5 mL PBMCs + 5 mL CFSE) in 15mL tube.
Place in 37°C H2O bath x 10 min.
Wash in 10 mL complete medium.
Resuspend in medium at desired density
