Difference between revisions of "Shreffler:Milk Program"
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Revision as of 11:48, 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
- 5,6-CFDA/SE Invitrogen C1157 dissolved in DMSO at 5 mM and stored at -20°C.
Isolation of Mononuclear Cells
CFSE labelling of 7day culture cells
- Suspend PBMCs at 10x106 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 H2O bath x 10 min.
- Wash in 10 mL complete medium.
- Resuspend in medium at desired density
- Lyons AB and Parish CR. Determination of lymphocyte division by flow cytometry. J Immunol Methods. 1994 May 2;171(1):131-7.
- 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 |
- 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 |
- Who has experience with this protocol?
- Email Wayne Shreffler through OpenWetWare