Difference between revisions of "Shreffler:CFSE Labeling"

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(Procedure)
(Undo revision 313541 by Wayne G. Shreffler (Talk))
 
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#Suspend PBMCs at 10x10<sup>6</sup> cells/mL in PBS alone.  
 
#Suspend PBMCs at 10x10<sup>6</sup> cells/mL in PBS alone.  
#Cells should be in PBS for CFSE labeling.
+
#Ensure that cells are uniformly suspended when CFSE is added.
 
#If CFSE negative control is needed, remove cells now.
 
#If CFSE negative control is needed, remove cells now.
 
#Make '2X' concentration (10 μM) in PBS
 
#Make '2X' concentration (10 μM) in PBS
 
#*For example: add 5 mL PBS + 10 μL 5 mM CFSE
 
#*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.
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#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.
 
#Place in 37°C H<sub>2</sub>O bath x 10 min.
 
#Wash in 10 mL complete medium.
 
#Wash in 10 mL complete medium.
#Resuspend in medium at desired density  
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#Resuspend in medium at desired density
  
==Notes==
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==Discussion==
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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[[Talk:{{PAGENAME}}|discuss this protocol]]
#List troubleshooting tips here. 
 
#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
 
#Anecdotal observations that might be of use to others can also be posted here. 
 
 
 
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
 
  
 
==References==
 
==References==
 
<biblio>
 
<biblio>
pmid=8176234
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#Lyons pmid=8176234
 
</biblio>
 
</biblio>
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 +
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==Contact==
 +
*Who has experience with this protocol?
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*[[Special:Emailuser/Wayne G. Shreffler|Email Wayne Shreffler through OpenWetWare]]
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[[Category:ShreffLab]]
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[[Category:Protocol]]
 
[[Category:Protocol]]
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[[Category:Flow cytometry]]
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[[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

  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
    • For example: add 5 mL PBS + 10 μL 5 mM CFSE
  5. Combine 1:1 PBMC cells + CFSE (e.g. 5 mL PBMCs + 5 mL CFSE) in 15mL tube and mix completely.
  6. Place in 37°C H2O bath x 10 min.
  7. Wash in 10 mL complete medium.
  8. Resuspend in medium at desired density

Discussion

discuss this protocol

References

  1. Lyons AB and Parish CR. Determination of lymphocyte division by flow cytometry. J Immunol Methods. 1994 May 2;171(1):131-7. PubMed ID:8176234 | HubMed [Lyons]


Contact