On the validity of fluorimetric intracellular calcium detection: Impact of lipid components

We investigated the effects of different lipids on the activity of the angiotensin II type 1 receptor (AT1R). As calcium plays a key role in the signaling of the AT1R, we used the calcium-sensitive fluorescence indicators fura-2 to detect intracellular calcium release upon stimulation with the agonist angiotensin II. At first sight, cells preincubated with Very low-density lipoprotein (VLDL) showed a reduced calcium release triggered by angiontensin II compared to untreated control. However, on closer examination, this result seemed to be an artifact. Incubation with VLDL reduced also the amount of intracellular fura-2, as measured by fluorescence in the isosbestic point. Additionally, the maximal obtainable ratio, obtained after complete saturation with calcium ions, was reduced in cells preincubated with VLDL. These findings rendered our initial results questionable. We report the results of our work and our suggestions regarding the experimental setup to contribute to the understanding of the interpretation of fura-2 measurements and to avoid erroneous conclusions. •Lipids may alter properties of cells with regard to fura-2.•Availability of intracellular calcium indicator fura-2 may be reduced.•If reporting of the fura-2 ratio only, key assumptions must be checked.•Ratio from saturated indicator (Rmax) should be verified.•Fluorescence at the isosbestic point (360 nm) should be verified.