Difference between revisions of "Shreffler:Milk Program"

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(Procedure)
(Procedure)
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'''Basophil Assay and Isolation of Mononuclear Cells''' (done simultaneously)
 
'''Basophil Assay and Isolation of Mononuclear Cells''' (done simultaneously)
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#Bring 15/10mL of Ficoll to room temperature and put 5mL RPMI in water bath. 
  
  

Revision as of 13:04, 27 July 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.


T regulatory Assay

Materials

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

Procedure

Basophil Assay and Isolation of Mononuclear Cells (done simultaneously)

  1. Bring 15/10mL of Ficoll to room temperature and put 5mL RPMI in water bath.


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

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]
  2. Fuss IJ, Kanof ME, Smith PD, and Zola H. Isolation of whole mononuclear cells from peripheral blood and cord blood. Curr Protoc Immunol. 2009 Apr;Chapter 7:Unit7.1. DOI:10.1002/0471142735.im0701s85 | PubMed ID:19347849 | HubMed [Fuss]
  3. Shreffler WG, Wanich N, Moloney M, Nowak-Wegrzyn A, and Sampson HA. Association of allergen-specific regulatory T cells with the onset of clinical tolerance to milk protein. J Allergy Clin Immunol. 2009 Jan;123(1):43-52.e7. DOI:10.1016/j.jaci.2008.09.051 | PubMed ID:19130927 | HubMed [Shreffler]
All Medline abstracts: PubMed | HubMed

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