Difference between revisions of "Shreffler:CFSE Labeling"

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==CSFE Labeling==
 
==CSFE Labeling==
  
Suspend PBMCs at 10x106 cells/mL in PBS alone. Cells must be in PBS for CFSE labeling.
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*Materials
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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] as 5 mM stock in DMSO
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*Procedure
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Suspend PBMCs at 10x10<sup>6</sup> cells/mL in PBS alone.  
 +
 
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Cells should be in PBS for CFSE labeling.
 +
 
 
If CFSE negative control is needed, remove cells now.
 
If CFSE negative control is needed, remove cells now.
Create solution of PBS:5μM CFSE
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CSFE is found in -20 degree fridge in 17-46.
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Make '2X' concentration (10 μM) in PBS
There are 2 CFSE stocks available - 40mM and 5mM.
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*Add 4mL PBS + 8 μL 5 mM CFSE (for example)
Want to start with 10μ M (“2X”).
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Add 4mL PBS + 8 μL 5 mM CFSE (for example)
 
 
Combine 1:1 PBMC cells + CFSE (i.e. 300uL PBMCs + 300uL CFSE) in 15 ml tube.
 
Combine 1:1 PBMC cells + CFSE (i.e. 300uL PBMCs + 300uL CFSE) in 15 ml tube.
 +
 
Place in H2O bath x 10 min.
 
Place in H2O bath x 10 min.
Wash in 10 mL AIM-V (10 min at 300G). Aspirate.
 
If plating...
 
resuspend in AIM-V to make 4x106 cells/mL (2X). There are now 2 million cells/ 0.5 mL.
 
Cells in tube are now all CFSE labeled and can be plated.
 
If CFSE positive control is needed, remove cells now.
 
If conducting Robocep selection, resuspend in Robocep buffer at appropriate concentration and follow protocol.
 
  
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Wash in 10 mL complete medium (10 min at 300G). Aspirate.
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Resuspend in medium at desired density
  
 
[[Category:Protocol]]
 
[[Category:Protocol]]

Revision as of 18:37, 19 May 2009

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CSFE Labeling

  • Materials

CFDA/SE Invitrogen C1157 as 5 mM stock in DMSO

  • 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

  • Add 4mL PBS + 8 μL 5 mM CFSE (for example)

Combine 1:1 PBMC cells + CFSE (i.e. 300uL PBMCs + 300uL CFSE) in 15 ml tube.

Place in H2O bath x 10 min.

Wash in 10 mL complete medium (10 min at 300G). Aspirate.

Resuspend in medium at desired density