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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===Materials===
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==Overview==
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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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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.
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===Procedure===
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==Materials==
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Suspend PBMCs at 10x10<sup>6</sup> cells/mL in PBS alone.  
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*15 mL polypropylene tubes
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*[[PBS]]
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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.
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Cells should be in PBS for CFSE labeling.
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==Procedure==
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If CFSE negative control is needed, remove cells now.
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#Suspend PBMCs at 10x10<sup>6</sup> cells/mL in PBS alone.
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#Ensure that cells are uniformly suspended when CFSE is added.
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#If CFSE negative control is needed, remove cells now.
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#Make '2X' concentration (10 μM) in PBS
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#*For example: add 5 mL PBS + 10 μL 5 mM CFSE
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#Combine 1:1 PBMC cells + CFSE (e.g. 5 mL PBMCs + 5 mL CFSE) in 15mL tube and mix completely.
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#Place in 37°C H<sub>2</sub>O bath x 10 min.
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#Wash in 10 mL complete medium.
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#Resuspend in medium at desired density
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Make '2X' concentration (10 μM) in PBS
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==Discussion==
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*Add 4mL PBS + 8 μL 5 mM CFSE (for example)
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[[Talk:{{PAGENAME}}|discuss this protocol]]
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Combine 1:1 PBMC cells + CFSE (i.e. 300uL PBMCs + 300uL CFSE) in 15 ml tube.
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==References==
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<biblio>
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#Lyons pmid=8176234
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</biblio>
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Place in H<sub>2</sub>O bath x 10 min.
 
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Wash in 10 mL complete medium (10 min at 300G). Aspirate.
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==Contact==
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*Who has experience with this protocol?
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*[[Special:Emailuser/Wayne G. Shreffler|Email Wayne Shreffler through OpenWetWare]]
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Resuspend in medium at desired density
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[[Category:ShreffLab]]
[[Category:Protocol]]
[[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

  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. . pmid:8176234. PubMed HubMed [Lyons]


Contact

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