Shreffler:CFSE Labeling

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==CSFE Labeling==
==CSFE Labeling==
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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.
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If CFSE negative control is needed, remove cells now.
If CFSE negative control is needed, remove cells now.
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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
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There are 2 CFSE stocks available - 40mM and 5mM.
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*Add 4mL PBS + 8 μL 5 mM CFSE (for example)
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Want to start with 10μ M (“2X”).
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Add 4mL PBS + 8 μL 5 mM CFSE (for example)
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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.
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Place in H2O bath x 10 min.
Place in H2O bath x 10 min.
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Wash in 10 mL AIM-V (10 min at 300G). Aspirate.
 
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If plating...
 
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resuspend in AIM-V to make 4x106 cells/mL (2X). There are now 2 million cells/ 0.5 mL.
 
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Cells in tube are now all CFSE labeled and can be plated.
 
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If CFSE positive control is needed, remove cells now.
 
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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 21:37, 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

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