Shreffler:CFSE Labeling

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===Materials===
===Materials===
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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.
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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
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*Add 4mL PBS + 8 μL 5 mM CFSE (for example)
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*Add 4 mL 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.
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Combine 1:1 PBMC cells + CFSE (e.g. 5 mL PBMCs + 5 mL CFSE) in 15mL tube.
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Place in H<sub>2</sub>O bath x 10 min.
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Place in 37°C H<sub>2</sub>O bath x 10 min.
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Wash in 10 mL complete medium (10 min at 300G). Aspirate.
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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 21:44, 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

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