Dear Akram,

problems like compartimentalization of Fluo-4 can be due to an excessive incubation time of the indicator. I usual incubate Hek cells with 1 uM fluo-4 for 30 minutes at 37°C followed by 20 minutes of de-esterification before imaging.

Moreover, the incubation solution must be composed by Fluo-4, sulfinpyrazone (final concentration 0.25 mM) and pluronic (final concentration 0.02% w/v). These two compounds are essential to avoid the dispersion of Fluo-4 in the acqueous medium and the leakage of the indicator from cells.

Good luck.

Thank you Salvatore,

I am wondering if you have ever done that with these compounds? Also, I am gonna treat the cells with ASM inhibitor to block calcium influx. Do you know any good inhibitor for that?

Thanks

Dear Akram

the concentration of fluo4 is high. try to use 100 uM of fluo4 AM and 200uM cell tracker.

Moreover, the loading period you used is much longer , should be 30 min. is enough and at dark room temperature to prevent photo bleaching of cells. then washing twice with DMEM or HS before imaging .

Hi Suha,

thanks for answer, My lab-mate is working with the same cell lines and the same concentration of Fluo-4 and he got good results. I think 100uM is pretty high for imaging by con-focal.

Hi Akram,

I always used those compounds in my experiments with Fluo-4 and I did not observe any problem.

I never used ASM inhibitor so I cannot say anything about that. In order to block calcium influx you have to study the channels that your cell model uses in order to uptake calcium. For example with cardiomyocytes nifedipine is a good inhibitor of calcium influx. In HEK cells maybe it would be enough to put the cells in 0 Ca2+ in the extracellular medium.

Thanks again,

Do I need to readjust the PH of media after adding these compounds?

Hello! Akram an apology but I had not seen your question

What you can intant is load with 10 μM 1 Hour at 37 degrees cetigrados of Fluo 4 -AM. We sometimes put PluronicF-127 a detergent that makes small pores and helps the dye enter.

we put 0.5% pluronic

Good luck

regards

Addition of Pluronic Acic at 0.5% is very useful to load ionic dyes into some cell types. Try it and tell us if it was useful to you!!

Regards,

Hi I have not still added Pluronic because my PI believes that adding a compound can not solve our problem for loading.

I tried another way and I did not wash after 1 hour and imaged the cells without washing. I could see a reasonable loading. I am not sure how necessary is washing for de-esterification?

I believe that washing excess of dye before the assay is essential to claim that the signal you are detecting is specific.

Regards,

De-esterification should happen in any case, with or without washing. The wash is essential to remove the excess of dye and to remove dead cells or cellular detritus in which the dye could accumulate giving you noise.

Hi akram

I used pluronic F 127 detergents with fluo4 am. Perhaps the washing with H.s should be twice after loading to have good imaging.

My labmates at prof ding's lab. work on Hek cell. You can contact with them I have already following them. Hou pan pan is my friend on research gate.

. You can read his papers to guide you for that.

Thank you Dear Suha, Could you tell me which objective have you used for confocal imaging?

The objective of microscope is 60 X oil immersion .

Hi Akram,

I don't know why you chose HEK 293 to do this experiment, since there is little calcium channel expression on the membrane of this cell line. If you want to observe the calcium influx in 293 cells, I suggest you to transfect a certein type of calcium channel (perhaps VGCC or LGCC) first which can induce Ca2+ release from ER. Moreover, 20 min is long enough for Fluo-4 AM incubation. Good Luck!

Thanks Wenyong

Hello Akram Etemadi I have recently purchased fluo-4am dye, can you please share the protocol for confocal imaging using this dye.

Thanks

I finally tried 2uM final concentration in media and incubate the cells for 30 min in 37C and wash them 2 times with media and incubate for 15min in 37C. I did not use any detergent to load Fluo-4 and used 10X objectives to get the signal. It depends on your experiment to use detergents to load better.

Hi Akram (and others on this chain) - I have a sort of similar question to yours. I am using Fluo-4 to look at the calcium gradient in fibroblasts (3T3 cells). But I want to image for an extended period (roughly 8 hours). Most protocols say that the cells need to be submerged in HBSS or some similar buffer during imaging but if cells remain in such buffers for a long time they will inevitably start dying. So I am thinking of submerging the cells in regular culture media (without the serum). Will I still be able to see the signal during fluorescence imaging? Or will any potential auto-fluorescence of the media affect the imaging?

Dear Appy Mukherjoo,

I suggest you to test for autofluorescence beforehand. If you’re using GFP, for example, set up the experiment under the scope with the cells but without the GFP. Illuminate with the GFP filter set, look for any visible fluorescence and adjust accordingly.

We usually add HEPES buffer in the media to decrease the PH changes affects.

I have never done hours live imaging but I think having a control for that might help you to find the best media.

Also look at this paper too.

Article Cell culture medium affects GFP photostability: A solution

Hi Akram Etemadi - thanks for your response! So when you treat your cells with the Fluo-4, you do you load the dye in HBSS (or HEPES) buffer solution and then add it to the culture and incubate it? After incubation, I am assuming you wash the cells a couple of times with HBSS (or HEPES)? After the washes do you again submerge the cells in fresh HEPES before you start imaging? Just trying to figure out what the protocol is because the journal papers are very brief in the methods section.

Thanks,

Appy

Appy, I do not use HBSS, I am using DMEM which has HEPEP (5ml in 500ml) and L-Glutamine (5ml in 500ml) all the process (incubating and washing) is done with the media.

I incubate the cells with fluo-4 (2ul in 1ml) for 30 min in 37C. then wash with media 2 times and rest for 30 min.

By the way, I am working with HEK293 cells. and my experiment is short time.

Akram Etemadi Thanks - that makes a lot of sense! I guess I will contact the company to figure out whether using regular cell media can mess with the fluorescence for long-term imaging (8 hours) or if they only recommend some colorless buffer like HBSS (in which case the cells may die over long periods of time). Thanks again for sharing!

Hi，Akram Etemadi，I have a same problem as yours. I wondered did you transfect 293 cells with some calcium channels? Or you succeed in loading fluo-4 AM for 293 cells without any transfection? Another question is that is there some calcium in DMEM media? And did these calcium ions interfere with the loading of Fluo-4 AM? Thank you very much!

I'm back to answer my own questions...There is no need to transfect 293 cells with calcium channels before loading Flou 4-AM, and the calcium ions in the media do not interfere with the loading process. I recommend the Fluo-4 AM produced by invitrogen. The dye could be successfully loaded either in HBSS solution at room temperature or in DMEM media without FBS at 37℃ for 40-60 mins.