Shreffler:Milk Program

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'''Isolation of Mononuclear Cells'''
'''Basophil Assay and Isolation of Mononuclear Cells''' (done simultaneously)

Revision as of 17:00, 27 July 2009



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.

T regulatory Assay


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


Basophil Assay and Isolation of Mononuclear Cells (done simultaneously)

CFSE labelling of 7day culture cells

  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

Proliferation Assay


discuss this protocol


Error fetching PMID 8176234:
Error fetching PMID 19347849:
Error fetching PMID 19130927:
  1. Error fetching PMID 8176234: [Lyons]
  2. Error fetching PMID 19347849: [Fuss]
  3. Error fetching PMID 19130927: [Shreffler]
All Medline abstracts: PubMed HubMed


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