Adrian Smith A.Smith at
Thu Oct 15 18:55:38 EST 1998

>Good afternoon!
>I really could use some help with a new application. I have an investigator
>who first stains 10 million mouse lymphocytes with CFSE (carboxyfluorescein
>diacetate succinimidyl ester dissolved in DMSO; final concentration = 5 ug
>CFSE per ml; 0.1% DMSO) in a total volume of 1 ml, washes the cells in
>PBS/10% FBS, and then stains for cell surface expression of CD4.
>It appears that we have a number of problems. First of all, treatment with
>CFSE drastically alters the light scatter measurements and CD4 expression
>appears to be lost. Secondly, CSFE staining is so bright that we have a
>difficult time compensating FL1 fluorescence out of FL2.
>Does any one have any experience with CFSE staining? Any suggestions?
>Thanks in advance,
>Julie Oughton
>Oregon State University

How soon after staining with CFSE do you try to run the cells? We find that
there is no way you can run the cells immediately after staining because
they are way too bright. This might account for the apparent loss of CD4
expression as well (if, for example you were staining with CD4PE, the PE
would be swamped by the CFSE). The cells need to be active (ie you can't
just leave them in the fridge) for at least 24 hours before you can expect
them to behave on the flow cytometer. Even then they are extremely bright
and you would need a very bright PE stain to be able to compensate
correctly. CFSE intensity drops dramatically in the first 24 hours of
metabolism and then slowly declines over the next several weeks (assuming
it is not diluted out by division of course). We haven't noticed that the
light scatter changes, but then again once we worked out they were too
bright to look at we haven't tried looking at them immediately after
staining. It is conceivable that they are changed soon after labelling but
they certainly return to "normal" after culturing (or transfer in vivo).

You could try reducing the concentration if you wanted to look at the cells
earlier - you would have to do a titration. This would of course reduce the
amount of time the cells will retain detectable label. (Our normal protocol
is 10 million cells/mL, 5uM CFSE, 10min staining at 37°C, in medium without
FCS, and then quencing the reaction with ice-cold medium with FCS).

We routinely stain for a wide range of surface markers (and intracellular
antigens) along with CFSE and as long as the CFSE intensity has decayed
sufficiently then we usally have no problems. To start with we had to stain
a large number of cells in compensation samples so we could fiddle with the
settings and get it right. Now we use the same settings files and they vary
very little from week to week.

If you need anymore help just let me know,

Adrian Smith

Adrian Smith (PhD Student)
Centenary Institute of Cancer Medicine & Cell Biology
Locked Bag No.6 Newtown, NSW 2042 AUSTRALIA.

Ph: 61-2-9565-6197 Fax: 61-2-9565-6103
A.Smith at
OR adriansmith at

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