Hi Julie,
I was cruising the list and read your question.
Sorry about the delay. If you're still considering JC-1. Here is my reply
to a previous question (and yours). (see link)
http://www.cyto.purdue.edu/hmarchiv/2000/1987.htm
why aren't untreated cells considered to be both orange and
green?
I always considered them as such.
Other tips:
JC-1 dilutions are sensitive to the buffer used. Solubility is an
issue. (don't have the data in front of me but believed we used DMSO as the
primary solvent). e.g. JC-1 diluted into a low-protein buffered salt
solution will "partition out" over time.
As stated in the link, a large amount of compensation is a must.
The degree of separation you get is based on your ability to use
compensation to amplify the difference between normal (red + green) and
uncoupled (green only).
The way I did this was to use an uncoupled (we had great results
with FCCP) and a normal control and maximize the differences between them
using compensation control. That's the only way I could get results as in
"figure 2". The compensation is very touchy and is dependent on JC-1
loading. Therefore you could not necessarily expect that the settings you
used for one experiment to be exactly applicable to the next.
It is not like the compensation you would do for immunophenotyping
(sorry if this is obvious), and it should not be considered quantitative for
delta psi. However, you can do some gating and collect data on % green
only, etc.
P.S. I could not see an attachment with your post. Feel free to send
directly.
Cheers,
Sam
Sam Witherspoon
sw11527@gsk.com
Mail Stop 3.2024A.2B
High Throughput Biology Tel. 919-483-3078
GlaxoSmithKline R&D Page 919-857-7768
5 Moore Dr. Fax 919-483-0585
RTP, NC 27709
-----Original Message-----
From: Oughton, Julie [SMTP:julie.oughton@orst.edu]
Sent: Thursday, May 09, 2002 3:21 PM
To: Cytometry Mailing List
Subject: JC-1
I just had a head-banging experience with JC-1 fluorescence. One of
my users
wants to examine the mitochondrial membrane potential of colon
epithelial
cells, using JC-1 [final concentration = 2.5 ug/ml].
We are using a Coulter XL flow cytometer; collecting data in FL1
[530
bandpass] vs FL-2 [575 BP]. We also collected data in FL1 vs FL-3
[620 BP].
Volts for each PMT was ~580.
Negative control: Untreated cells
Positive control: Cells treated with 0.1mM valinomycin
in DMSO
for 2 hours
I am finding it very difficult to compensate JC-1 fluorescence.
According to
archived messages on this list, compensation can be quite tricky.
>From what
I understand, the positive control should be producing green
fluorescence
while untreated cells mostly orange. According to the fluorescence
pattern
in figure 2 at:
http://flowcyt.cyto.purdue.edu/flowcyt/research/cytotech/amfc/data/page13.ht
m
why aren't untreated cells considered to be both orange and green?
Unfortunately, the pattern of our JC-1 fluorescence (monomers vs
aggregates)
looks quite bizarre [see attached histogram, no compensation]. In
addition,
valinomycin treatment resulted in a large population of cells that
exhibited
low FS [see attached figure; these cells are color evented as red],
probably
apoptotic cells. And their fluorescence pattern was not what we
expected.
I would love to discuss our data with someone who has experience
with JC-1.
Any suggestions to help set proper compensation? Any comments?
Thanks in advance!
Julie Oughton
Oregon State University
> -----Original Message-----
> From: Oughton, Julie [SMTP:julie.oughton@orst.edu]
> Sent: Thursday, May 09, 2002 3:21 PM
> To: Cytometry Mailing List
> Subject: JC-1
>
>
>
> I just had a head-banging experience with JC-1 fluorescence. One of my
> users
> wants to examine the mitochondrial membrane potential of colon epithelial
> cells, using JC-1 [final concentration = 2.5 ug/ml].
>
> We are using a Coulter XL flow cytometer; collecting data in FL1 [530
> bandpass] vs FL-2 [575 BP]. We also collected data in FL1 vs FL-3 [620
> BP].
> Volts for each PMT was ~580.
>
> Negative control: Untreated cells
> Positive control: Cells treated with 0.1mM valinomycin in DMSO
> for 2 hours
>
> I am finding it very difficult to compensate JC-1 fluorescence. According
> to
> archived messages on this list, compensation can be quite tricky. From
> what
> I understand, the positive control should be producing green fluorescence
> while untreated cells mostly orange. According to the fluorescence pattern
> in figure 2 at:
>
> http://flowcyt.cyto.purdue.edu/flowcyt/research/cytotech/amfc/data/page13.
> ht
> m
> why aren't untreated cells considered to be both orange and green?
>
> Unfortunately, the pattern of our JC-1 fluorescence (monomers vs
> aggregates)
> looks quite bizarre [see attached histogram, no compensation]. In
> addition,
> valinomycin treatment resulted in a large population of cells that
> exhibited
> low FS [see attached figure; these cells are color evented as red],
> probably
> apoptotic cells. And their fluorescence pattern was not what we expected.
>
> I would love to discuss our data with someone who has experience with
> JC-1.
> Any suggestions to help set proper compensation? Any comments?
>
> Thanks in advance!
> Julie Oughton
> Oregon State University
>
>
>
>
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