Antwort: simultaneous screening of 2 cell lines

Rafael Nunez nunezr at mskcc.org
Fri Mar 22 12:19:39 EST 2002


Hi Richard and Flowers, in Current Protocols in Cytometry of last
year, I published a protocol (9.17.9-11) for "assessment of in vivo
cytotoxicity by flow cytometry" by  labeling two populations of cells
with same dye but at different concentration and assess the cell
viability after in vivo killing of the targets cells. The reagents
are from Molecular Probes and is a very reliable technique. Then you
can add an antibody PE  labeled.  Regards Rafael
>I suspect that one might be able to do the following:
>
>Separately label the cell populations with calcein AM (or CellTracker Green
>CMFDA) but to different levels of brightness by using more or less of the
>tracer (or leave one population unlabeled). Both will have green fluorescence
>but one should be able to get at least a 10-fold difference in the
>intensities.
>The calcein will leak out of dead cells (a bit less likely in the
>CMFDA-stained
>cells) so it is preferable to label both cell lines. As a dead cell stain I
>would also recommend the green fluorescent SYTOX Green stain. Its much greater
>intensity in dead cells should make it easy to distinguish dead
>cells from live
>cells even in the presence of other green-fluorescent labels. Of course, one
>would have to balance the brightness of both green-fluorescent stains to get
>complete discrimination. Alternatively 7-AAD may be suitable for dead cells.
>For antigen discrimination a simple R-PE conjugate should then suffice.
>
>Currently there is not a particularly good tracer that can be detected in the
>far red with 488 nm excitation, although DiD (DiIC18(5)), which is
>structurally
>analogous to DiC18(3) (which is the structure of PKH26) may be bright enough
>even when excited at 488 nm with emission beyond 650 nm.
>
>
>eug.kreysch at t-online.de wrote:
>
>>  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 at Phylos.com am 15.03.2002 23:10:56
>>
>>  An:   cytometry at 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

--
Rafael Nunez M.D.
Associate Laboratory Member-Immunology Program.
Manager Flow Cytometry Core Facility.
Memorial Sloan-Kettering Cancer Center
Box 98, 1275 York Avenue.
New York, NY 10021. USA
Telephone (212) 639-6392
Fax (917) 432-2333
E-mail: nunezr at mskcc.org



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