Heidi,
The Coulter support person was correct. You can use 7-AAD to
discriminate live from dead cells. The excitation maximum is 550 nm and
the emission maximum is around 660 nm. This allows you to use a FITC or
FITC-like fluorochrome and PE simultaneously with the 7-AAD. See the
following reference:
SchmidI, Krall WJ, Uittenbogaart CH, Braun J, Giorgi JV: Dead cell
discrimination with 7-amino-actinmycin D in combination with dual color
immunofluorescence in singlelaser flow cytometry. Cytometry 13:204-8, 1992
hengel@APS.UoGuelph.CA on 11-Jan-1999 12:10 PM
To: cyto-inbox
cc: (bcc: Thomas J Williams)
Subject: (Fwd) 'alternative' live/dead stains?
Hello! I would like to pick the brains of the group for help in setting
up a three colour protocol. So far, my experience is
limited to double staining, using FITC and PE. I have had no
official training on the flow cyt, so there are huge gaps in my
knowledge ...
I am trying to set up a flow cytometric assay to detect lysis and
conjugate formation between target cells (K562 myeloid leukemia
cell line) and effector cells (natural killer (NK)-like lymphocytes
from the pregnant pig uterus). Uterine cells will be a mixed
population, with only a proportion of them lytic.
Another complication is that the uterine lymphocytes likely to be
mediating the killing are much larger than conventional NK cells.
Normally, one can distinguish targets from circulating NK cells by
size, but I suspect that my uterine effectors will be similar to the
target cells in size and granularity.
SO. I need to set up a three colour protocol. I need to label my
targets, label the dead cells, and somehow identify the effector cells
(surface phenotype - they strongly express CD16). Originally I was
considering the following strategy:
DiOc - to label targets (behaves much like FITC)
PI - to label deads
and a third label, conjugated to the secondary antibody I use to detect
the CD16-positive cells.
For this third label, I was thinking about 'Tri-Color' (TC; Caltag).
This combination looked good on paper (DiOc, PI, TC) - the TC
would be excited by the argon laser and emit at a sufficiently high
wavelength to be distinguished from the other two. However, I
discussed this with the Coulter flow cytometry support person, and
she warned me that PI has a very broad emission that interferes
with just about anything other than molecules emitting in the FITC
range.
She told me about an alternative to PI for live/dead staining - called
7-aminoactinomycin-D (7-AAD). I have been through the Molecular
Probes catalogue. It seems that 7-AAD is OK for cell cycle
analysis but not so good for viability. Does anyone have any
experience with this? There was a comment in the catalogue
about 7-AAD staining non-differentiated versus differentiated cells.
Staining of viable target cells (tumour cells) would make this stain
useless for our purposes ... (we are ordering the reference for this -
could be that because they were assessing DNA staining rather
than viability, they actually permeabilized the cells first).
Can anyone suggest alternative strategies?
Thanks!
Heidi Engelhardt
Heidi Engelhardt
Department of Animal and Poultry Science
University of Guelph
Guelph, Ontario
CANADA N1G 2W1
phone: 519-824-4120, ext. 3658 or 8355
fax: 519-767-0573
email: hengel@aps.uoguelph.ca
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