Difference between revisions of "Shreffler:CFSE Labeling"

From OpenWetWare
Jump to: navigation, search
(Undo revision 313541 by Wayne G. Shreffler (Talk))
 
(18 intermediate revisions by 2 users not shown)
Line 1: Line 1:
===Materials===
+
==Overview==
  
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.
+
CFSE is used to fluorescently label live cells in order to track them. 5,6-CFDA/SE is membrane permeable, but trapped in the cell when converted by intracellular esterases. CFSE is equally partitioned to daughter cells during division and can be used to measure cell proliferation.
  
===Procedure===
+
==Materials==
  
Suspend PBMCs at 10x10<sup>6</sup> cells/mL in PBS alone.  
+
*15 mL polypropylene tubes
 +
*[[PBS]]
 +
*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.
  
Cells should be in PBS for CFSE labeling.
+
==Procedure==
  
If CFSE negative control is needed, remove cells now.
+
#Suspend PBMCs at 10x10<sup>6</sup> cells/mL in PBS alone.
 +
#Ensure that cells are uniformly suspended when CFSE is added.
 +
#If CFSE negative control is needed, remove cells now.
 +
#Make '2X' concentration (10 μM) in PBS
 +
#*For example: add 5 mL PBS + 10 μL 5 mM CFSE
 +
#Combine 1:1 PBMC cells + CFSE (e.g. 5 mL PBMCs + 5 mL CFSE) in 15mL tube and mix completely.
 +
#Place in 37°C H<sub>2</sub>O bath x 10 min.
 +
#Wash in 10 mL complete medium.
 +
#Resuspend in medium at desired density
  
Make '2X' concentration (10 μM) in PBS
+
==Discussion==
*Add 4 mL PBS + 8 μL 5 mM CFSE (for example)
+
[[Talk:{{PAGENAME}}|discuss this protocol]]
  
Combine 1:1 PBMC cells + CFSE (e.g. 5 mL PBMCs + 5 mL CFSE) in 15mL tube.
+
==References==
 +
<biblio>
 +
#Lyons pmid=8176234
 +
</biblio>
  
Place in 37°C H<sub>2</sub>O bath x 10 min.
 
  
Wash in 10 mL complete medium.
+
==Contact==
 +
*Who has experience with this protocol?
 +
*[[Special:Emailuser/Wayne G. Shreffler|Email Wayne Shreffler through OpenWetWare]]
  
Resuspend in medium at desired density
+
[[Category:ShreffLab]]
  
 
[[Category:Protocol]]
 
[[Category:Protocol]]
 +
 +
[[Category:Flow cytometry]]
 +
 +
[[Category:Immunology]]

Latest revision as of 11:23, 10 June 2009

Overview

CFSE is used to fluorescently label live cells in order to track them. 5,6-CFDA/SE is membrane permeable, but trapped in the cell when converted by intracellular esterases. CFSE is equally partitioned to daughter cells during division and can be used to measure cell proliferation.

Materials

  • 15 mL polypropylene tubes
  • PBS
  • 5,6-CFDA/SE Invitrogen C1157 dissolved in DMSO at 5 mM and stored at -20°C.

Procedure

  1. Suspend PBMCs at 10x106 cells/mL in PBS alone.
  2. Ensure that cells are uniformly suspended when CFSE is added.
  3. If CFSE negative control is needed, remove cells now.
  4. Make '2X' concentration (10 μM) in PBS
    • For example: add 5 mL PBS + 10 μL 5 mM CFSE
  5. Combine 1:1 PBMC cells + CFSE (e.g. 5 mL PBMCs + 5 mL CFSE) in 15mL tube and mix completely.
  6. Place in 37°C H2O bath x 10 min.
  7. Wash in 10 mL complete medium.
  8. Resuspend in medium at desired density

Discussion

discuss this protocol

References

  1. Lyons AB and Parish CR. Determination of lymphocyte division by flow cytometry. J Immunol Methods. 1994 May 2;171(1):131-7. PubMed ID:8176234 | HubMed [Lyons]


Contact