Laird,
you could possibly use green fluorescent PKH2-GL (FL1) or red fluorescent
PKH26-GL (Sigma) (FL2) for membrane labeling, respectively, and 7-AAD for
dead cell exclusion in FL3. Thus, FL1 or FL2 could be used for detection of
antibody binding.
Best regards
Georg
LBloom@Phylos.com am 15.03.2002 23:10:56
An: cytometry@flowcyt.cyto.purdue.edu
Kopie:
Thema: simultaneous screening of 2 cell lines
Hi,
I'm looking for a method to label two cell lines differentially
so
that they can be mixed and then tested together for binding to an antibody
sample and for viability. The two cell lines differ only by the surface
antigen to which the antibodies are directed, so the differential labeling
cannot use a surface antigen. My cytometer is a FACSCalibur, with
standard
optics (488 nm excitation, and detection filters FL1=530/30, FL2=585/42,
and
FL3=650LP).
A recent paper (Yang, E-P, et. al. 2002. BioTechniques
32:678-682)
used the dyes CMFDA and CMTMR (Molecular Probes) for differential cell
labeling in the FL1 and FL2 channels, and a surface antigen was detected
with a Cy-chrome fluorophore (BD) in FL3. Live/dead discrimination was not
attempted in this paper, and the difference in FL3 signal between positive
and negative cell lines was not very large.
Presumably a cell line labeled with a single dye could be mixed
with an unlabeled line to achieve the same purpose as using the two dyes,
leaving one channel available for live/dead discrimination. Does anyone
have
an idea for a good combination of fluorophores that would allow me to do
this with minimal bleedthrough? It is particularly important that the
surface antigen fluorophore is well separated from potential bleedthrough
from the other two dyes, since it will most likely be the dimmest of the
three signals.
Would the following be reasonable: Alexa 488 in FL1 for the
surface
antigen, CMTMR in FL2, and 7-AAD in FL3? What about CMFDA or BCECF in FL1,
PE in FL2, and 7-AAD in FL3? Are there any good generic cell dyes for FL3?
Any suggestions would be welcome.
Thanks,
Laird Bloom
Phylos, Inc.
128 Spring St.
Lexington, MA 02421
tel. (781) 862-6400 ext. 253
fax (781) 402-8813
www.phylos.com
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:59:29 EST