Hi folks,
I'm cleaning out emails, and realized I never posted a summary of the
replies to my original question re: apoptosis induction for positive
controls. Thanks to all those who responded!
My original query:
Hi folks,
I have a user who is just getting started w/ apoptosis in a fibrosarcoma
cell line (HT1080). Does anyone have a best guess for a reliable positive
control treatment (camptothecin, UV, etc?). All our tests have been
negative to date, but we can't say it's not occurring unless we verify our
assay. (We're using PI at this point, although I'm trying to talk them
into "graduating" to tunel or annexinV.)
Thanks in advance,
Steve
And the numerous replys below:
From: "WAGNER, CONSTANCE A [R&D/1000]" <constance.a.wagner@pharmacia.com>
To: cyto-inbox
Subject: RE: apoptosis induction?
Hi Steve,
Campothecin works very well with HL-60 cells, but I have no experience
with
your cell line. I have not been in this line of work for about 5 years,
but
back then, I used a kit from Oncor, called Apoptag, and it worked pretty
well. I used this to verify what I saw with PI. I don't even know if
Oncor
is still in business, but it might be worth a try.
-----------
From: Andrew Beernink <ABeernink@novasite.com>
To: cyto-inbox
Subject: RE: apoptosis induction?
Ouabain or Actinomycin D should work, though I don't remember dosing or
incubation time (low micromolar to high nanomolar concentrations ring a
bell, ideally overnight). Hope that helps!
-----------
Robert Archer, Ph.D.
Cell Biology Product Manager
mailto:robert@probes.com
Molecular Probes, Inc.
Compound Final Concentration Solvent
Actinomycin D 0.5 µg/mL Methanol
Aphidicolin 2 µg/mL DMSO
A23187 10 mM DMSO
Caffeine 16 mM H2O
Camptothecin
(Topoisomerase I inhibitor) 4 to 100 µg/mL DMSO
Cycloheximide 100 µg/mL H2O
Dexamethasone 1 to 100µM Ethanol
Doxorubicin (adriamycin) 0.2 µg/mL H2O
etoposide 50 µg/ml
5-Fluorouracil 25 µg/mL
Hydroxyurea 2.5 mM PBS
Staurosporine 0.5 µM DMSO
Taxol (paclitaxel) 100 to 580 nM DMSO
Thymidine 2 mM PBS
Vinblastine 60 nM Methanol
-----------
From: sian rizzo <srizzo@sghms.ac.uk>
To: cyto-inbox
Subject: Re: : apoptosis induction?
Try 7AAD for apoptosis detection, lovely separation of
live/apop/dead, and it can be used with 2 other colours,
sometimes if flops into FL1, so if you have a machine that
you can't compensate FL3 out of FL1 it may not be suitable
to use FL1.
-----------
From: "nigel miller (BI)" <nigel.miller@bbsrc.ac.uk>
To: cyto-inbox
Subject: RE: apoptosis induction?
Hi Steve
I hear that referees of apoptosis papers are now demanding at
least
three methods to identify dying cells. I suggest your user tries
1.PI
2.FITC AnnexinV
3.Caspase
4.Mitotracker
5.Poss ChromaTide BODIPY FL-14-dUTP or SYBR Green 1 Gel Stain
6.Vybrant Apoptosis Assay Kit 4
------------
From: Dennis J. Young <djyoung@ucsd.edu>
To: cyto-inbox
Subject: Re: apoptosis induction?
I wouldn't even think of using TUNEL or even plain old PI until you see
DEAD cells by microscopy. Then you can assume they MAY have died by
apoptosis. I've seen a similar line HT116 that will apoptose with H2O2.
I'd
try the camptothecin.
------------
From: Daniel Chipchase <D.Chipchase@oxfordbiomedica.co.uk>
To: cyto-inbox
Subject: RE: apoptosis induction?
I'd appreciate it if you posted any other replies you get to the mailing
list.
I am using transfected TE671 and HT1080 and a FITC conjugated secondary
to detect surface expression of my protein of interest. Instead of using
PI I'm excluding dead cells (and apoptotic cells) using TO-PRO 3 which
is viewed in FL4 and thus allows you to use another fluorochrome in FL2.
It also means there is little or no compensation necessary.
For positives I simply expose cells to 70% Ethanol for >5mins, and then
stain along with all my other samples. 3 populations are usually visible
(similarly to PI), with an intermediate population thought to correspond
to apoptotic cells.
You could also use Hoechst 33342, nuclear uptake of which is blocked by
apoptotic cells, but not by dead cells.
Anyway to answer your question I believe Camptothecin treated cells may
be used as a positive control
http://www.promega.com/pnotes/57/5573b/5573b.html
has a good section on flow analysis of apoptotic cells, and lots of
references too.
Hope this helps.
-------------
--
+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
Steve G. Hilliard flowman@uga.edu
Flow Cytometry Facility 542-9474
University of Georgia
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