Re: PKH dyes
From: William Telford (TelfordW@mail.nih.gov)
Date: Mon Nov 26 2001 - 00:41:14 EST
Hi Tom...
The PKH dyes do have a tendency to transfer between cells over time -
I've posted some data at...
http://home.ncifcrf.gov/ccr/flowcore/PKH_transfer.pdf
The first page shows mixtures of unlabeled and PKH-labeled
fibroblasts (2 uM labeling concentration, 5 minutes labeling time)
co-cultured over eight days in serum-free conditions (to prevent cell
division-related dye loss). The least lipophilic dye (PKH2) starts
to transfer between cells by five days, while the more lipophilic PKH67
shows only minimal transfer after eight days. The second page shows
a longer comparison of the red-emitting PKH26 and PKH67, which have
similar levels of lipophilicity - significant transfer begins to occur
after 12 days. While PKH26 and PKH67 would therefore seem to be the
obvious choice for labeling, the tradeoff is that we see greater
inhibitory effects on cell proliferation than with PKH2 in both cell
lines and primary immune cell cultures (probably because of their longer
lipophilic tails and subsequent interference with membrane mobility and
proteins). So they may indeed be altering membrane protein
epitopes. Like CFSEDA, therefore, the best way to use these dyes is
to titer them down as much as possible for your particular
application. They are all extremely bright, so you may be able to
use them at 1 uM or less, depending on your application. Reducing
the labeling time also reduces the dye incorporation levels - we can go
as low as one or two minutes and still get bright labeling.
The PKH dyes also have a tendency to label intracellular membranes, such
as the ER. Using the lowest concentration and labeling time you can
get away with reduces this problem as well.
Good luck,
Bill
At 09:13 AM 11/21/2001 -0500, Ahern, Thomas P wrote:
Greetings,
I'm currently using CFSE to indiscriminantly stain tumor cells. I'm concerned that the reaction with free amine groups may be altering some unique tumor-specific epitopes on the membrane surface (which I'm trying to target with cominatorial peptide libraries).
Is PKH more amenable to preserving the antigenic architecture of surface proteins? I understand it uses two lipophilic 'anchor' regions which insert into the bilayer, and hence may sterically hinder access to some epitopes without actually altering their structure. Are there any problems with diffusion of this dye from cell to cell?
Regards,
Tom.
Thomas Ahern
Vermont Cancer Center
E-315 Given Building
Burlington, VT 05405
(802) 656-2218 voice
thomas.ahern@uvm.edu
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