cell blebbing with flou4-AM
kavitha thangaraj
Friday, 13 March 2009 06:18 UTC
Im Doing my Phd. currently working on live cell imaging.
I’m using fluo4-am (ca indicator, final concentration 5micromolar)it is dissolved in DMSO. As soon as i add the fluo4 to the cells they started blebbing (necrosis?).
i tried with DMSO alone. the cells (L6, skeletal myoblast) look quite good with DMSO.they are blebbing only with fluo4 dye. I’m also using hoechst dye to stain nucleus. i incubate them for 30 min. de-esterification is for 20 min.
can any one answer for my question?
thanks in advance.
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Hi Kavitha,
Is your dye dissolved in 20% Pluronic acid as well?
I might have a few suggestions:
-you could try to decrease the final concentration of Fluo4, even down to 1 micromolar
-the loading time can also be decreased (15-20 minutes) but apparently the cells start dying right away
-it might be useful to test another batch of Fluo4 or try Fluo3 which has similar properties to thoses of Fluo4
-you could try to use another batch of L6 cells. Do you have identical problems when you load other types of cells with Fluo4?
Are your cells already differentiated? Is there a difference in the blebbing phenomenon before and after differentiation?Hope this helps a bit.
Good luck,
Cedric.
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Hi Cedric,
thanks for the response. i don’t use pluronic acid. i need to think about it. do you think 1 micromolar concentration dye is enough to stain cells in 35mm dishes? can u tell me the stability of fluo4-AM. how long it can stay with good intensity. do you think using hoechst is advisable?once again thanks for your valuable suggestions.
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Hi Kavitha,
-1 micromolar Fluo4 could be enough but obviously it depends on the density of the cells and on the camera you have in your set-up. You could first try 4 micromolar, then 3 and 2, and finally work out the right concentration for your system.
-With Fluo4, you might encounter the problem of anti-bleaching (meaning the opposite of photobleaching). Indeed, you might observe an increase of the basal fluorescence level due to the fact that some fluorophores acquire the ability to permanently fluoresce. It’s a bit tricky to handle this issue but I think if you reduce the power of the laser and reduce the exposure time, it should be all right.
-I think I’ve missed the point with the Hoechst staining… So you perform a double labeling of your cells, right? Does the blebbing phenomenon also appear when you only load the cells with Fluo4? By the way, Fluo4 itself does a good staining of the nuclei in many types of cells, if it’s what you’re looking at. It is due to a difference in the apparent Ca-binding between cytoplasmic and nucleoplasmic regions.
-For more information (especially on photobleaching and intensity), you might find the following article useful:
A comparison of fluorescent Ca2+ indicator properties and their use in measuring elementary and global Ca2+ signals. Thomas D, Tovey SC, Collins TJ, Bootman MD, Berridge MJ, Lipp P. Cell Calcium. 2000 Oct;28(4):213-23.Best wishes,
Cedric.
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Hi Cedric
thanks for the valuable suggestions. its working good with 4micromolar concentration. thanks for the reference article. -
Hi Cedric and kavitha,
I have a question on Fluo-4 am too and I am wondering if you guys can shed some light on it. I use Fluo-4 am on myofibroblasts and it seems my cells are taking the dye well (very bright fluorescence signal when seeing under FITC). However, when I tried to add KCl to stimulate intracellular Ca2+ spike, there is no change on the fluorescent intensity at all, at least I can’t see any change with my naked eye. I use dye concentration at about 3-4 uM with Ploronic.I’ve also tried my cells with Fura-2 on a ratiometric system, the system can pick up Ca2+ changes after stimulating with KcL, this means my cells are responding to the stimulus. I don’t know if the change with Fluo-4 is detectable with naked eye or it has to be detected using very sensitive camera. The camera I’m using can detect the fluorescence signal well at 500ms exposure time, but this seems overly long as compared with the ratiometric system (60ms exposure time).
Any input is welcome, thanks in advance.
Ruogang
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Hi Ruogang
are u using permeabilised myofibroblast? what is the concentration of KCL you are using? is the dye is bleaching? if ur dye is bleaching then u cannot identify the increase in fluorescence with time lapse. I think Cedric can give some better idea to u.best wishes.
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Hi Kavitha and Ruogang,
As far as I can tell, I think both of you are right concerning the apparent absence of KCl response in fluo4-loaded myofibroblasts.
Your exposure time (500 ms) is probably too long and I fear you get this anti-bleaching effect (basically the opposite of photobleaching, see reply of 25 Mar 2009). Moreover if you see a very bright fluorescence signal when seeing under FITC, it’s unfortunately a good indication that your cells are over loaded with fluorphores that permanently fluoresce and are not “reactive” any more. As I proposed to Kavitha (see again reply of 25 Mar 2009), you might try to reduce the intensity of the laser, certainly try to reduce the exposure time (if possible down to 100 ms or even less), and try to reduce both the loading time (30 minutes might be too much, you could maybe go down to 15 min or something in between) and the dye concentration. You seem to have a bright signal for 3 micromolar fluo4, so I would suggest to test 1 or 2 micromolar with your cells. After the loading and the de-esterification process, the fluorescence signal of your cells should be very faint, so that you can hardly see the cells under FITC.Hope this helps.
Cedric
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Hi Kavitha,
My cells are not permeabilized for Ca2+ study, they are intact. I’m using high concentration KCL at 0.5-1M. Yes, the Fluo-4 am dye bleaches. According to some literature, Fluo-4 am dye supposes to bleach very little, but on my system, apparent fluorescent intensity decrease can be observed after 30 seconds if I leave the shutter open.I read some articles saying that Ca2+ fluorescence images should not be observed using eyes, otherwise it will bleach fast.
Another reason I can think of is the focus drift. Since when I add the stimulus liquid in, the focal plane changes and this causes the fluorescence to decrease drastically. During this period, the Ca2+ transient change has probably passed.
Thanks for the input.
Ruogang
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Hi Cedric,
Just see your reply, great suggestions! I think my cells are definitely over loaded so that they are not responding any more! I’ll try to reduce the dye concentation and loading time. I’ll let you guys know how they response.Thanks very much.
Ruogang
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Hi Cedric and kavitha,
I followed your suggestions and used low dye concentration (1-1.5uM) and less incubation time (20 min). My cells now look more like Cedric described: very faint under FITC, which indicates a correct loading condition. I also reduced the exposure time to 60ms. However, my cells still not respond to the stimulus very much. The percentage of cells responded to either KCL and ATP stimulation has been very low. Actually, I have only seen and captured with video the fluorescence increase for less than 5 cells in my last several trials. Is this normal that only very small population of cells will respond to stimulation?One reason I can think of is the loading media. I’ve been using MEM-alpha as working media for dye loading; however, it seems most of the publications have used HEPEs buffered saline as working media. I guess the osmolarity probably plays an important role here. Have you experience any problem with this?
Thanks,
Ruogang
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