Hello everyone,
For those interested in the effects of permeabilization reagents on light
scatter properties of paraformaldehyde-fixed cells, the following reference
looks at Saponin, Triton-X and ethanol.
Lecoeur, H., E. Ledru, M-L. Gougeon. 1998. A cytofluorometric method for
the simultaneous detection of both intracellular and surface antigens of
apoptotic peripheral lymphocytes. J. Imm. Meth., 217:11-26.
There was also a group in Europe that were assessing many of the
commercially available fixation/permeabilization reagents for their
effectiveness in staining several CD and disease important epitopes, but I have
not seen that work published as yet. Hope this adds something to the discussion.
-Jerry Wilson.
Flow Cytometry Reagent Development
Biological Response Modifier group
BD Biosciences- San Diego.
"Jayaraman, Sundararajan" <SJayaram@med.miami.edu> on 11/23/99 08:45:34 AM
To: cyto-inbox
cc: (bcc: Jerry Wilson/SDCA/BDX)
Subject: RE: Intraprep reagent & light scatter
Sarah,
I fully agree with Randy Fischer about the changes in scatter properties
after fixation/permeabilization. I have used BD's Lysis solution and
permeabilization solution to determine IL-2. IL-4, and IFN-gamma
intracellularly in cultured human T cells. I found significant reduction in
the forward scatter after permeabilization. This is true in the human T cell
leukemia cell line, Jurkat also.
Jay
Sundararajan Jayaraman, Ph.D.
Research Assistant Professor
Dept. of Pathology
University of Miami Medical School
DRI Building Room 5018 (R-134)
1450 NW 10th Avenue
Miami, FL 33136
Phone: (305) 243-6100
Fax: (305) 243-4553
> -----Original Message-----
> From: Fischer, Randy [SMTP:RFischer@therimmune.com]
> Sent: Friday, November 19, 1999 10:20 AM
> To: Cytometry Mailing List
> Subject: RE: Intraprep reagent & light scatter
>
>
> Sarah,
>
> A helpful point about intracellular staining is that in general all the
> fix/permeabilization procedures available, either commercial or home
> brew, will cause a change in the scatter properties of the cells.
> Slight adjustments in your settings on the controls I know you run will
> bring back your populations and this is perfectly acceptable.
>
> Have fun,
>
> Randy T. Fischer
> TherImmune Research Corporation
> 9700 Great Seneca Hwy
> Rockville, MD 20850
> (240) 453-6256
> RFischer@therimmune.com
>
> > ----------
> > From: Sarah Lawson
> > Sent: Saturday, November 13, 1999 5:33 AM
> > To: Cytometry Mailing List
> > Subject: Re:Intraprep reagent & light scatter
> >
> > Dear All,
> >
> > I am relatively new to flow cytometry and have a query regarding
> > Coulter Immunotech Intraprep reagent. Has anyone got any experience
> > with this product? We are finding a quite marked change in scatter
> > properties following its use, although we can obtain the same scatter
> > pattern by increasing FS and SS total gain. Does this matter?
> >
> > Any comments would be gratefully received.
> >
> > Kind regards,
> >
> > Sarah Lawson
> > ( isaac.lawson@btinternet.com)
> >
> >
> >
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