Shreffler:CFSE Labeling: Difference between revisions

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(New page: {Shreffler} ==CSFE Labeling== Suspend PBMCs at 10x106 cells/mL in PBS alone. Cells must be in PBS for CFSE labeling. If CFSE negative control is needed, remove cells now. Create solution...)
 
(Undo revision 313541 by Wayne G. Shreffler (Talk))
 
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{Shreffler}
==Overview==


==CSFE Labeling==
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.


Suspend PBMCs at 10x106 cells/mL in PBS alone. Cells must be in PBS for CFSE labeling.
==Materials==
If CFSE negative control is needed, remove cells now.
 
Create solution of PBS:5μM CFSE
*15 mL polypropylene tubes
CSFE is found in -20 degree fridge in 17-46.
*[[PBS]]
There are 2 CFSE stocks available - 40mM and 5mM.
*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.
Want to start with 10μ M (“2X”).
 
Add 4mL PBS + 8 μL 5 mM CFSE (for example)
==Procedure==
Combine 1:1 PBMC cells + CFSE (i.e. 300uL PBMCs + 300uL CFSE) in 15 ml tube.
 
Place in H2O bath x 10 min.
#Suspend PBMCs at 10x10<sup>6</sup> cells/mL in PBS alone.  
Wash in 10 mL AIM-V (10 min at 300G). Aspirate.
#Ensure that cells are uniformly suspended when CFSE is added.
If plating...
#If CFSE negative control is needed, remove cells now.
resuspend in AIM-V to make 4x106 cells/mL (2X). There are now 2 million cells/ 0.5 mL.
#Make '2X' concentration (10 μM) in PBS
Cells in tube are now all CFSE labeled and can be plated.
#*For example: add 5 mL PBS + 10 μL 5 mM CFSE
If CFSE positive control is needed, remove cells now.
#Combine 1:1 PBMC cells + CFSE (e.g. 5 mL PBMCs + 5 mL CFSE) in 15mL tube and mix completely.
If conducting Robocep selection, resuspend in Robocep buffer at appropriate concentration and follow protocol.
#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
 
==Discussion==
[[Talk:{{PAGENAME}}|discuss this protocol]]
 
==References==
<biblio>
#Lyons pmid=8176234
</biblio>
 
 
==Contact==
*Who has experience with this protocol?
*[[Special:Emailuser/Wayne G. Shreffler|Email Wayne Shreffler through OpenWetWare]]
 
[[Category:ShreffLab]]
 
[[Category:Protocol]]
 
[[Category:Flow cytometry]]
 
[[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. DOI:10.1016/0022-1759(94)90236-4 | PubMed ID:8176234 | HubMed [Lyons]


Contact

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