James N. Mittler
mittler at wadsworth.org
Thu Oct 15 15:02:08 EST 1998
I have been using CFSE to monitor the cell division of antigen-specific CD4
cells in vivo. I stain the cells at 5e7 cells/ml PBS (no FBS), the CFSE at
a final concentration of 5uM, for 10 min. at 37C. Following staining I do
3 washes with RPMI/10%FBS. Surface staining and light scatter are not
affected by staining in this manner.
CFSE is very bright. I have found it impossible to compensate the FL2
channel. I have had success using fluorochromes such as Red670 and
TriColor (FL3 on FACScan) for surface staining.
I hope this helps.
- JIM M. -
At 04:30 PM 10/13/98, Julie Oughton wrote:
>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,
>Oregon State University
James N. Mittler
University at Albany
School of Public Health
Department of Biomedical Sciences
Laboratory of Immunology
David Axelrod Institute
<mittler at wadsworth.org>
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