Shreffler:Milk Program

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

Isolation of Mononuclear Cells

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. DOI:10.1016/0022-1759(94)90236-4 |
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 7:7.1.1-7.1.8. DOI:10.1002/0471142735.im0701s85 |
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 |

Contact

• Who has experience with this protocol?
• Email Wayne Shreffler through OpenWetWare