CFSE staining of T cells
Make a 107 cells/ml cell suspension in PBS, BSA 0.1%. I do the staining in a 50ml falcon tube.
If necessary keep an aliquot for FACS.
Add CFSE (from a 10mM stock solution) at a final concentration of 1-10μM depending on the cell type used. For T cells generally between 5μM and 10μM are just right.
NOTE: The CFSE stock solutions are kept at -20oC, in small 20μl alicots that are defrosted, used and thrown away. I always put in the box that contains the alicots, some of these granules that catch water because the CFSE is sensitive to humidity.
Keep 10 min at 37oC in the water bath.
If you want to have a stronger staining you can play with the CFSE concentration and the time, and add a couple of more minutes for the staining. But do not exceed 15min.
Add an excess of complete medium FCS 10% RPMI to wash. Complete to 50ml.
Spin at 1500rpm / 5min.
Resuspend the cells in complete medium.
Count the cells again, usually there is 50% loss in the numbers, so do not be surprised.
Spin at 1500rpm / 5min.
Resuspend the cells in RPMI alone, to 2×106/200ul (–> 10×106/ml).
Cells are ready for iv injection.
CFSE Staining of lymphocytes —> PDF