I understand JC-1 staining is used by many people to monitor mitochondrial membrane potential in apoptosis induction in a variety of cell systems. I see also different ways of interpreting the results (so I am still not persuaded). I'd like to know how you interpret the findings in JC-1 staining, I mean, what is the best and widely accepted way? (whether analysis in FACS, IF or a plate reader).
Are you monitoring J-aggregate and monomers using 2 sets of excitation/emission wavelengths? The most common and accepted analysis is to divide J-aggregate fluorescence by monomer fluorescence, or vice versa. You should see the ratio change using an uncoupler control, such as dinitrophenol, FCCP, or CCCP.
Why are you not persuaded? Do you see no change in your experiments?
Hi Gin:
Thanks for the reply, that's actually the point!, vice versa? or only the former!. I know that JC-1 is actually a ratio-metric assay. I do see nice changes, but some times both mono and aggr. hugely decrease (FACS and plate reader), basically we expect increase in monomers but always decrease in aggr.(am I right?). Under the FL microscope I see a lot of green stained cells (monomers) and red (aggregates) at the same time even in untreated cells. With other apoptosis-related markers and assays that I've done, I am highly convinced.
You're correct then, especially in seeing untreated cells showing both green and red, while depolarized cells will show less red. I don't think it matters whether you calculate green/red or red/green.
JC-1 is a ratio-metric assay. This sounds quite easy but may include many unknown parameters. Usually, people use flowcytometers to calculate the ratio of red:green stained cells after treatment or only for culture condition analysis. But in my opinion people measure this assay with a flowcyt caused by historically reasons– former they hadn’t had cell imagers!
I set up the JC-1 assay for my cell imager and I figured out, the most sufficient and stable way is to choose this JC-1 concentration and incubation time which makes a green fluorescence in all cells (treated and untreated) and only the red fluorescence should decrease with a rising conc. of toxin or what else… And then you should always calculate the red:green ratio!
What I’ve seen in the past is, the people often use too high JC-1 concentrations which causes red J-aggregates (concentration depending) in all (apoptotic) cells, which leads in confusing results! And in my opinion without the images (like it is the issue with flowcyometers) I would never have found the error in my experiments!!
For my cell line (Nalm-6; pre-B-Lymphocytes) a concentration of 0.5 µM over 30’ @37° C is ideal.
Hi Tina,
I use a Leica microscope imager and tested 2uM JC-1 for 6-10min on human fetal astrocytes in large tilescans. First time testing I had aggregates of the dye, will test filtering the final solution before incubating. How do you determine the red/green ratio in images? I mechanicall stretch-traumatize cells, so some cells are injured others are not.
thanks
Ina
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