Posted by
Kilgore, Jason-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Deconvolving-Spinning-Disk-Images-tp6508446p6510344.html
** Vendor Response **
Hi, Michal,
The dyes that are SE (succinimidyl ester) derivatives will bind to proteins (via their lysine residues) in the cytoplasm, covalently, and thus are retained long-term. 3-4 days is not a problem with these. CFDA SE is the most commonly used. These are fixable due to that protein label and crosslinking with aldehydes.
Most of the CellTracker dyes also will bind to proteins as well, via thiol groups, and be retained over at least 4 days as well (with the exception of CellTracker BODIPY and CellTracker CM-DiI, which are lipophilic membrane stains). They are also fixable, as with the SE dyes.
I would recommend avoiding the lipophilic cyanine dyes, including the PKH kits, due to the fact that they just intercalate into the membranes without covalently binding to cell constituents, and are not as fixable.
Another choice is to use Qdots, namely our Qtracker cell labeling reagents, which are actively taken up by endocytosis and sequestered into endosomes. These, too, are retained with fixation (and can even survive paraffin processing).
It looks like you have filter options for a number of these choices.
If you would like more help in choosing, please let me know, offline.
Cheers,
Jason
Jason A. Kilgore
Technical Application Scientist
Molecular Probes Labeling and Detection Technologies
Cells Systems Division
T 1 800 955 6288 then option 5 or 541 335 0353 • F 541 335 0238
29851 Willow Creek Rd • Eugene • OR • 97402-9132 • United States
www.invitrogen.com/technicalsupport
-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of Michal Opas
Sent: Thursday, June 23, 2011 2:39 PM
To:
[hidden email]
Subject: Live fixable fluorescent cell labelling
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Hello Dear Listers,
We have run into a conundrum regarding live, semi-long term and fixable
labelling of two population of mouse ES cells for aggregation/sorting
out studies.
We would like to fix label cells (two cell strains), let them aggregate,
fix the aggregates after 3-4 days and be able to make them out with either
am ancient Bio-Rad MRC 600 Krypton/Argon (488, 568 and 647 nm) or
a prehistorical Zeiss Photomicroscope (DAPI/Hoechst @ ~350 nm, FITC @
~390 nm and TRITC (~440 nm) ot Texas Red (~620 nm).
Invitrogen/Molecular Probes
(
http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Fluorescent-Tracers-of-Cell-Morphology-and-Fluid-Flow/Membrane-Permeant-Reactive-Tracers.html#head2)
offer quite few cell tracking probes but figuring out what does what is
difficult (for me, at least).
They say that CellTracker Green CMFDA (5-chloromethylfluorescein
diacetate) labels cells that are both fluorescent and viable for at
least 24 hours after loading.
We need three-four days, though.
Orange-fluorescent CellTracker Orange CMRA - ditto.
CellTrace Oregon Green 488 Carboxylic Acid Diacetate Succinimidyl Ester
is highly recommended but nothing is said of its fixability.
It is said that CellTrace Far Red DDAO-SE is a fixable,
far-red–fluorescent tracer for long-term cell labelling but from the
literature given I am not sure of its longevity.
Sigma
(
http://www.sigmaaldrich.com/img/assets/8200/PKH_LinkerKit_web_version.pdf)
sells PKH26 and PKH67 that (Sigma says) are stable for up to 100 days in
vivo (PKH26), but I do not see them heavily used in cell tracking other
than FACS.
/Ergo/,
Any practical suggestion shall be greatly appreciated!
Greetings,
Michal
PS
Obviously (and to my chagrin) I have no financial association with
neither company.
M.O.