CFSE and cell division Part II

Adrian Smith A.Smith at centenary.usyd.edu.au
Thu Jun 18 22:22:37 EST 1998


>Derek,
>
>This procedure has been very successful in our hands and we now have
>combined it
>with Apoptosis and CD25-PE.  Please note that these are only possible with the
>use of a green HeNe and gated amp because the CFSE activation contaminates
>most
>of your PMT's.

It is certainly possible to use other fluorochromes with CFSE. We have used
up to four other colours with CFSE on a Facstar Plus (PE, Texas Red, APC,
and PI or PerCP). However compensation can be difficult. It helps to have a
bright PE reagent and then to turn down the FITC  (so there is not so much
spill over into the PE channel). Plenty of time and losts of single colour
sample is also useful... (alternatively you can try post-aquisition
software compensation, for example in FlowJo).
CFSE is far too bright for use on the cytometer immediately after labelling
(at least at the normal concentrations). There is rapid loss of intensity
over the first 24 hours (but not if the cells are at 4°C) and then a slower
loss over the next few weeks. Presumably this is related to the turn over
of intracellular proteins that have been conjugated to the dye. Of couse
once division starts the intensity of the divided cells decreases two-fold
with each division. It is matter of balancing how bright you want your
intial staining to be (and hence how many divisions you can distinguish),
how long you before you want to analyse your cells, and the toxicity of the
staining process (at high concentrations more cells are lost during
labelling). For most purpose 5-10uM seems the best.

On a more embarassing note, the basis of the problem with the concentration
has been revealed. One of the papers I referenced before (Hasbold, J., A.
B. Lyons, et al. (1998). "Cell division number regulates IgG1 and IgE
switching of B cells following stimulation by CD40 ligand and IL-4." EJI
28: 1040-1051.) incorrectly states the concentration of CFSE used in nM
rather than uM. Oops....I think (hope) the rest of the papers I referenced
are right.

Adrian Smith


******************************************************
Adrian Smith (PhD Student)
T CELL BIOLOGY GROUP
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 centenary.usyd.edu.au
******************************************************





More information about the Cytometry mailing list