Shreffler:CFSE Labeling
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Current revision (14:23, 10 June 2009) (view source) (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. | ||
| - | # | + | #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. | + | #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 | + | #Resuspend in medium at desired density |
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| + | ==Discussion== | ||
[[Talk:{{PAGENAME}}|discuss this protocol]] | [[Talk:{{PAGENAME}}|discuss this protocol]] | ||
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#Lyons pmid=8176234 | #Lyons pmid=8176234 | ||
</biblio> | </biblio> | ||
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==Contact== | ==Contact== | ||
*Who has experience with this protocol? | *Who has experience with this protocol? | ||
| + | *[[Special:Emailuser/Wayne G. Shreffler|Email Wayne Shreffler through OpenWetWare]] | ||
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| + | [[Category:ShreffLab]] | ||
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| + | [[Category:Protocol]] | ||
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| + | [[Category:Flow cytometry]] | ||
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| + | [[Category:Immunology]] | ||
Current revision
Contents |
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. . pmid:8176234.
Contact
- Who has experience with this protocol?
- Email Wayne Shreffler through OpenWetWare
