-I am using Fura-2AM dye with probenecid to permiabilise TR146 oral epethelial cells.
-flourescence is detected on a Flexstation.
-Results from current experiments vary significaltly to previously collected data and there is also a greater level of variability between biological repeats.
-The only things I can think of that may be a cause are:
the quality/age of the Fura-2 (it was reconstituted about a year ago and has been stored at -20
user error
issues with the cells used
If anyone has any other suggestions it would be greatly appreciated!
Hi Rhys,
My lab has done a lot of work with Fura2 over the years, so I thought I'd message to see if we can help. First thing though, you mention that you are using probenecid to permeabilise your epithelial cells. Probenecid doesn't actually permeabilise cells (at least, not to my knowledge). People use probenecid to stop cells extruding Fura2 (some cell types are very good at doing this; especially if the experimental temperature is increased above 20oC). We have used probenecid to stop Fura2 extrusion, but we find sulphinpyrazone an order of magnitude more effective.
Is the Fura2 loading in your cells consistent? What temperature are you loading the cells with the Fura2AM and for how long? Can you use a local wide field imaging system with 340 nm or 380 nm excitation to make sure that your Fura2 is loading well and is clearly cytosolic? You can check that it's cytosolic by trying a mild permeabilization of your cells using a low concentration of digitonin- this will release Fura2 from the cytosol but leave behind any Fura2 that is trapped in organelles. It's always a good idea to check the loading of cells with fluorescent indicators at the single cell level before scaling up to population experiments.
The Fura2 you are using is a little old, and I'd be tempted to make a fresh stock for experiments, but it might be ok. Again, I would test this at the single cell level by imaging the response of cells to a generic calcium-mobilising agonist such as ATP.
Other issues could arise from the cells themselves. For example, is the cell passage number or density the same between experiments? How are you stimulating your cells to show calcium signals? Is it a robust response (albeit variable in your experiments)? Finally, how are you quantitating your responses? I'm presuming you are using background-corrected 340/380 ratio measurements?
Apologies if these thoughts/suggestions are not helpful. As I mentioned, I'd recommend doing an investigation of how your cells are responding at the single cell level to ensure the Fura2 loading is ideal, that the cells are healthy and not heterogeneously responsive to your stimulus. Once you have established protocols that work at the single cell level, all should be fine for scaling up to population measurements on a plate reader.
Best of success with your experiments.
Martin
Hi Rhys,
My lab has done a lot of work with Fura2 over the years, so I thought I'd message to see if we can help. First thing though, you mention that you are using probenecid to permeabilise your epithelial cells. Probenecid doesn't actually permeabilise cells (at least, not to my knowledge). People use probenecid to stop cells extruding Fura2 (some cell types are very good at doing this; especially if the experimental temperature is increased above 20oC). We have used probenecid to stop Fura2 extrusion, but we find sulphinpyrazone an order of magnitude more effective.
Is the Fura2 loading in your cells consistent? What temperature are you loading the cells with the Fura2AM and for how long? Can you use a local wide field imaging system with 340 nm or 380 nm excitation to make sure that your Fura2 is loading well and is clearly cytosolic? You can check that it's cytosolic by trying a mild permeabilization of your cells using a low concentration of digitonin- this will release Fura2 from the cytosol but leave behind any Fura2 that is trapped in organelles. It's always a good idea to check the loading of cells with fluorescent indicators at the single cell level before scaling up to population experiments.
The Fura2 you are using is a little old, and I'd be tempted to make a fresh stock for experiments, but it might be ok. Again, I would test this at the single cell level by imaging the response of cells to a generic calcium-mobilising agonist such as ATP.
Other issues could arise from the cells themselves. For example, is the cell passage number or density the same between experiments? How are you stimulating your cells to show calcium signals? Is it a robust response (albeit variable in your experiments)? Finally, how are you quantitating your responses? I'm presuming you are using background-corrected 340/380 ratio measurements?
Apologies if these thoughts/suggestions are not helpful. As I mentioned, I'd recommend doing an investigation of how your cells are responding at the single cell level to ensure the Fura2 loading is ideal, that the cells are healthy and not heterogeneously responsive to your stimulus. Once you have established protocols that work at the single cell level, all should be fine for scaling up to population measurements on a plate reader.
Best of success with your experiments.
Martin
Hi Martin,
Firstly, thank you very much for such a long a detailed response, It is much appreciated!
Also, thank you for the clarification on the function of probenecid and the alternative, sulphinpyrazone.
in answer to your questions, we are loading the same molar concentration of Fura in every experiment. The internalisation of the dye is done at 37.5 C. We use ionomycin treatment to induce a maximal response and we use a second positive control- candidalysin. this is a fungal toxin that previously been shown to induce calcium influx.
your suggestions regarding wide field imaging are very useful. As the protocol was optimised by someone else in our lab I will speak to them and find out of this was done.
these results are a little odd as I have just completed another project which utilised this technique and the data was very consistent, and the controls looked as expected.
like you said the Fura 2 was a little old I will make up more (when I can get back to the lab...). the cells were a moderatly high passage but not so high that I would expect encounter issues. However, I guess I can't really rule out that these are the issue until I use a fresh batch!
Again, thank you fo taking the time to asnwer my question! all the suggestions, hints and tips are very useful. Rest assured im taking lots of notes!!
All the best in these strange and difficult times.
Rhys Brown
Mr. Brown,
You need mass spectrometry for such analysis, instead of FS method, due to limitations of the latter method for quantitative purposes.
Please, consider the following discussion and the reference section therein:
https://www.researchgate.net/post/ELI5_FRET_assay_Protease_assay_Michelis_Menten_How_would_you_convert_the_fluorescence_signal_to_the_velocity_concentrion_time
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Techniques
- Blotting Techniques
- Immunoblotting
- Dyes