CFSE

To study proliferation, CFSE stains membranes and is diluited 1:2 at each cell division step

Pellet 20*10⁶ cells and wash wash with 10mL PBS (you can go down to 5*10⁶cells)

centrifuge

Resuspend in PBS 20*10⁶/1mL PBS

Add 1:1 diluited CFSE (CFSE 2µM, e.g. 0.5µL stock (40mM in DMSO) in 10mL PBS) so to have a final staining conc of 1µM

Leave 8’RT, after 4’ mix by hand

Block staining by adding an equal volume of FBS

Leave 1’RT

Centrifuge

Wash with 5mL complete medium and leave 5’RT

Centrifuge

Resuspend and count (have you lost cells? CFSE may be toxic) and plate (you may do an additional wash step)

Acquire an unstimulated control staining (you can also fix cells)

The unstimulated population should be between 10^4-10^5