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.
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===Procedure===
+
==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
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Cells should be in PBS for CFSE labeling.
+
==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.
-
If CFSE negative control is needed, remove cells now.
+
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
-
 
+
-
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 H<sub>2</sub>O bath x 10 min.
+
-
 
+
-
Wash in 10 mL complete medium.
+
-
 
+
-
Resuspend in medium at desired density
+
 +
==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

  1. Suspend PBMCs at 10x106 cells/mL in PBS alone.
  2. Cells should be in PBS for CFSE labeling.
  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.
  6. Place in 37°C H2O bath x 10 min.
  7. Wash in 10 mL complete medium.
  8. Resuspend in medium at desired density

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. 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

  1. =8176234 [pmid]
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