Thanks to all who helped me with JC-1 fluorescence.
We have initiated studies to look at several options. First, we changed our
filters to collect orange fluorescence, using a 590 nm BP filter. Secondly,
the investigator used staurosporine, rather than valinomycin, with much more
favorable results. My investigator is also looking into the use of TMRE
[Molecular Probes].
Here is a summary of all the replies that I received.
Thanks again!
Julie Oughton
Oregon State University
********************************************************************
This is my original post:
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?
****************************************************
And these are the replies:
**************************************************
My feeling on this is with JC-1 you don't really want to try to set
compensation. You can't anyway because you can't do anything to the cells
to get pure green or pure orange fluorescence. Then you have to consider why
you would want to in the first place. You usually set compensation when two
different dyes are independently reporting on two different markers. In this
case the same dye, JC-1, is reporting in both channels. Your looking for a
shift from green to orange. In my experience I've never actually seen an
increase in orange, just a decrease in green. At any rate, the greater the
decrease in green (and if you've got it, the greater the increase in orange)
the more apoptosis you have. You'll see this just as well no matter what you
do to the compensation settings and whatever conclusions you can draw with
compensation you can draw without it.
********************************************************
You may want to try an excellent dye for determining the inner mitochondrial
potential disruption. TMRE sold by Molecular Probes ( no endorsement) is
very bright and no problem of compensation. Simply incubate (1 million
cells/ml) in complete tissue culture media along with 40 nM of TMRE for 20
min at 37oC. Wash once in staining buffer (Dulbecco's PBS + 0.2% BSA, filter
sterilized; azide is not required if cells are analyzed within hours) and
resuspend in the same buffer and keep the cells on ice till you run on a
flow cytometer. I generally can get away with 100,000 cells per condition. I
have used a variety of human cell lines as well as primary cells. There are
a number of reports using TMRE out there. You may also want to check the
website of Molecular Probes for full details and references. Good luck.
************************************************
First, I should introduce myself. I'm Dax Arguello, and I'm the Beckman
Coulter instrument sales rep for your area. I work directly with Dave
Osborn.
Funny you should ask about JC-1. I was just working the ISAC meeting and a
customer brought up the same issue. It was another customer from Colorado
who mentioned to me how much better results they've gotten using the
Advanced Digital Compensation they just upgraded to on their XL. I would
suspect the way ADC calculates the compensation algorithm helped in her
case.
I can talk to Karen and see what they're doing to make it work, because I've
seen many problems with people trying to use JC-1. I actually will be
meeting with her next week. If that's too far down the road, I'll see if I
can get her on the phone before that time.
Please let either Dave or me know how else we can help.
********************************************
I have used JC1 a lot. It is at best, a dye which leaves a lot to be
desired. It requires a ton of epifluorescent microcopy. It would not
recommend staining your cells and running flow. You really need to look
at these under the fluorescent scope to understand what you are getting
from the flow. Compensation is different for each cell type. As you have
seen, the data is confusing and difficult to understand and if one is
not examing the prep's under the fluorescent scope, forget it. Also,
remember JC1 is very temperature sensitive!
***************************************************
I have done JC-1 on an XL configured as is yours and neither FL2 nor
FL3 is really optimal for the fluorescence of the J-aggregates in intact
mitochondria. One simple solution would be to start removing some
filters and replacing with empty filter holders. I believe the peak of
the J-aggregates is about 595nm so maybe removing the FL2 filter would
be a place to begin. Probably the best solution would be to put the 645
dichroic where the 600 is now located (or just remove the 600 dichroic)
and then put a 585 long pass filter in front of the PMT in which you
want to collect the orange fluorescence. As you likely know, the
voltages have to be turned WAY down from typical immunofluorescence
settings.
The normal cells are in fact both green and orange, at least the ones
I have examined. I don't believe that valinomycin will collapse the
mitochondrial potential. I used it pre-JC-1 as a hyperpolarization
control with the cyanine dyes. Perhaps PMA or staurosporine, or other
agents that induce apoptosis would be useful for your cells. With
neutrophils, I know that PMA essentially instantly wipes out the orange
JC-1 fluorescence.
The other dye from Molecular Probes may also solve your problems.
Good luck with the studies.
*********************************************************
First, yes, JC-1 J-aggregate form is Orange and Green together . . .
conversion to monomer form is seen as a loss of Orange with a concomitant
increase in Green. A fully depolarized sample will show all Green,so that's
what you must do to get the Green-only control for compensation.
Probably what you're seeing here is a viability issue . . . probably a
valinomycin dose-specific effect. Look a viability following valinomycin
treatment. Minimize your val dose to a level that still shows the
conversion (you can easily monitor via fluorescence microscopy).
**************************************************************
Unfortunately I didi not received your histograms attached to your mail from
the cytometry mailing list. But I can tell you how we measure apoptotic T
cells with JC-1.
After the incubation for 48h in the presence of 10 mM 2-deoxy-D-ribose and
harvesting I stain the CD3 cells with the CD3-APC antibody from BD. To avoid
the toxic effects of Sodiumazid I dialysed the antibody before.
After a few minutes I add the JC-1, vortex immediately and stain the cells
for 15 minutes. Then I wash and measure them.
The measurement is done here with an FACSCalibur and its advantage is that
JC-1 is only weakly detecteable in the fourth channel. I do not make any
compensation, so JC-1 is detected in FL1, FL2 and FL3. But with the staining
of CD3-APC I can exclude all the rubbish and the cells I am not interested
in.
For the analysis I make a dotplot JC-1 (green) against JC-1 (orange) and
define the cells in the strong orange and light green area as vital and the
rest as apoptotic/dead. In the attached dotplot you can see the vital cells
in the blue ellipse. The cells are pregated on CD3+!
I think the big problem is that you do not have a stainable marker (or is
there any?) to separate your cells from the rest.
To give you an idea I would do it like this:
Start with the normal settings, reduce the voltage in FL1 and FL2 (JC-1 is a
very bright color). Decrease the compensation FL1 against FL2 and vice versa
to zero and control the FL4 channel by single stained cells in the case that
you have an specific antibody coupled to PerCP or PECy5 for your cells. And
then compare your untreated cells with the valinomycin treated cells. If you
want to stain JC-1 and antibody together be sure that the antibody is free
of Sodiumazid otherwise you can see nearly no cells with an intact
H+potential over the mitochondrial membrane.
***************************************************
two suggestions:
1) don't attempt to compensate - turn all compensation off.
2) look at FL1 (530 +/- 20) and FL3 (>650nm) and use ratios
anybody attempting to compensate using a single fluorochrome doesn't
understanstand what compensation is for.
**************************************************************
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.
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