Why is my fluorescence intensity excited at 340 and 380 following the same trend?

23 February 2016 7 7K Report

I am trying for the first time to mesure Calcium influx in stimulated/unstimulated Basophils using Fura-2 AM (incubation for 30min at 37degrees) and a plate reader. I was hoping that when I get high values for 340nm, the intesity values for 380 have to decrease, but this in not the case. When 340 goes up, also the 380 does. Is this normal? What am I doing wrong?

Thank you!

Ada Yeste

Thank you Peter for your answer!

How can I do a good calibration? Thank you again!!!!!

Thomas Launey

Hi Ada,

You may have a look at https://biofrontiers.colorado.edu/core-facilities/microscopy-core/user-resources-1/protocol-for-staining-cells-with-fura-2 for a quick protocol and link. Hope this helps.

Ada Yeste

Thank you Thomas! Your link was very helpful!

Anthony J Morgan

Hi Ada,

We use Ca2+ dyes a lot, and I'm afraid I would be really sceptical that your signals = Ca2+ changes.

With no disrespect to the people above, the ratio is NOT the be all and end all. You must be mindful of the raw data to eliminate potential artefacts.

As a ratiometric dye, it must show anti-parallel changes at each wavelength (or at least the 380 should decrease while the 340 stays the same/increases). Seeing the signals change in the same direction is pretty diagnostic of an artefact.

Are you sure you have enough fura-2 in the cells? Check this by the Mn2+ quench technique at the end of the run. Your F2 signal should be at least 5-times the background autofluorescence. If it isn't, your cells are not loaded enough.

(1) is crucial because cells autofluoresce in the UV range. Indeed, people measure cell autofluorescence to monitor dynamic NAD(P)H changes e.g. Ca2+ can stimulate mitochondrial metabolism, and the increase in reduced pyridine nucleotides manifests itself as an increase in autofluorescence at both these wavelengths (what you see !).

You might be getting cellular pH changes that affect the dye (it is H+ and not Ca2+ that is changing).

Changes in cell shape or movement ? Also if your cells are in suspension (as I presume basophils are) do they float to the bottom of the well ? if so does addition of drug merely waft more cells into the light path?

Can you see a robust anti-parallel response with a strong stimulus e.g. ionomycin ?

Some plate reader optics are really poor at monitoring fura-2 (depends on the configuration and filter sets). Check with an in vitro calibration in an intracellular-like buffer containing fura-2 (K+ salt, NOT AM-ester); have one without Ca2+ (Rmin + 10mM EGTA) and one with saturating Ca2+ (e.g. 1mM Ca2+). This will give you the dynamic range of the machine and inform you as to how good the optics are. You will also be able to judge the brightness of your 'loaded' cells (see (2)) and the dye in solution.

Some cells show dye compartmentation (the AM ester is cleaved inside organelles, not just the cytosol). This can also screw up your interpretation.l Image your cells to check for punctate/reticular staining with F2; also try permeabilizing the plasma membrane and see how much dye exits from the cytosol (should be all of it; if most of it is left behind, it is inside organelles where Ca2+ can be high/behave differently from the cytosolic signal).

Hope this helps.

Best,

Anthony

Marie-Louise Ward

As Anthony says!

Santosh Yadav

Hi Anthony,

Anthony J Morgan

Thanks for such an elaborated answer with an explanation.

I have a follow-up question from your answer as you mentioned the 380 should decrease while the 340 stays the same. I have the same effect where 380 decreased but 340 remain the same with one of the treatments of the compound in platelets what does it mean?

Thanks,

Santosh

Guilherme Henrique Souza Bomfim

Hi Ada,

As the others colleagues highlighted, your issue could be related to low efficiency of Fura-loaded cells as well as in the plate reader capacity to detect changes in the Ca2+-free and bound ratio. Try to calibrate the plate reader and check the probe saturation point, maybe can be useful.

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