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

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 |

Contact

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