Shreffler:CFSE Labeling: Difference between revisions
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== | ==Overview== | ||
5,6-CFDA/SE | 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 [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. | |||
==Procedure== | |||
If CFSE negative control is needed, remove cells now. | #Suspend PBMCs at 10x10<sup>6</sup> 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 H<sub>2</sub>O bath x 10 min. | |||
#Wash in 10 mL complete medium. | |||
#Resuspend in medium at desired density | |||
==Discussion== | |||
[[Talk:{{PAGENAME}}|discuss this protocol]] | |||
==References== | |||
<biblio> | |||
#Lyons pmid=8176234 | |||
</biblio> | |||
==Contact== | |||
*Who has experience with this protocol? | |||
*[[Special:Emailuser/Wayne G. Shreffler|Email Wayne Shreffler through OpenWetWare]] | |||
[[Category:ShreffLab]] | |||
[[Category:Protocol]] | [[Category:Protocol]] | ||
[[Category:Flow cytometry]] | |||
[[Category:Immunology]] |
Latest revision as of 11:23, 10 June 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.
- 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
Discussion
References
- Lyons AB and Parish CR. Determination of lymphocyte division by flow cytometry. J Immunol Methods. 1994 May 2;171(1):131-7. DOI:10.1016/0022-1759(94)90236-4 |
Contact
- Who has experience with this protocol?
- Email Wayne Shreffler through OpenWetWare