Shreffler:CFSE Labeling
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*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. | ||
| - | + | ==Procedure== | |
| - | 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. | ||
| + | #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. | ||
| + | #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 | ||
| - | + | ==Notes== | |
| + | Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! | ||
| + | #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. | |
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| - | * | + | |
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| + | ==References== | ||
| + | <biblio> | ||
| + | pmid=8176234 | ||
| + | </biblio> | ||
[[Category:Protocol]] | [[Category:Protocol]] | ||
Revision as of 22:03, 19 May 2009
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.
- Cells should be in PBS for CFSE labeling.
- 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.
- Place in 37°C H2O bath x 10 min.
- Wash in 10 mL complete medium.
- Resuspend in medium at desired density
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- 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 '''*~~~~''': to the beginning of your tip.
References
- =8176234
