Shreffler:CFSE Labeling: Difference between revisions

Revision as of 18:37, 19 May 2009

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CFDA/SE Invitrogen C1157 as 5 mM stock in DMSO

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

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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 ==CSFE Labeling==
-Suspend PBMCs at 10x106 cells/mL in PBS alone. Cells must be in PBS for CFSE labeling.
+*Materials
+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
+*Procedure
+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.
-Create solution of PBS:5μM CFSE
-CSFE is found in -20 degree fridge in 17-46.
+Make '2X' concentration (10 μM) in PBS
-There are 2 CFSE stocks available - 40mM and 5mM.
+*Add 4mL PBS + 8 μL 5 mM CFSE (for example)
-Want to start with 10μ M (“2X”).
-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 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.
+Wash in 10 mL complete medium (10 min at 300G). Aspirate.
+Resuspend in medium at desired density
 [[Category:Protocol]]