Difference between revisions of "Shreffler:CFSE Labeling"

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===Materials===
 
===Materials===
  
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.
+
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.
  
 
===Procedure===
 
===Procedure===
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Make '2X' concentration (10 μM) in PBS
 
Make '2X' concentration (10 μM) in PBS
*Add 4mL PBS + 8 μL 5 mM CFSE (for example)
+
*Add 4 mL 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 (e.g. 5 mL PBMCs + 5 mL CFSE) in 15mL tube.
  
Place in H<sub>2</sub>O bath x 10 min.
+
Place in 37°C H<sub>2</sub>O bath x 10 min.
  
Wash in 10 mL complete medium (10 min at 300G). Aspirate.
+
Wash in 10 mL complete medium.
  
 
Resuspend in medium at desired density  
 
Resuspend in medium at desired density  
  
 
[[Category:Protocol]]
 
[[Category:Protocol]]

Revision as of 17:44, 19 May 2009

Materials

5,6-CFDA/SE Invitrogen C1157 dissolved in DMSO at 5 mM and stored at -20°C.

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

Combine 1:1 PBMC cells + CFSE (e.g. 5 mL PBMCs + 5 mL CFSE) in 15mL tube.

Place in 37°C H2O bath x 10 min.

Wash in 10 mL complete medium.

Resuspend in medium at desired density