Can I use a plate reader to measure the pH of acidic organelles, such as lysosomes, with LysoSensor ratiometric probes?
Using a plate reader for measuring the pH of acidic organelles, such as lysosomes, with LysoSensor ratiometric probes, provides weak pH measurements, as discrete regions of interest cannot be selected for analysis. Therefore, there is no way of excluding extracellular fluorescence and little to no spatial or morphological information can be obtained. Instead, a readout of whole-cell fluorescence is provided, resulting in the average intraorganellar pH of all labeled organelles for the analyzed cell population. The is also the risk that a signal may be dominated by a few extremely bright, but essentially irrelevant cells, such as dead cells, or dye-labeled debris particles.
For more information about measuring luminal pH in organelles, please see the reference linked below:
Live-cell Microscopy and Fluorescence-based Measurement of Luminal pH in Intracellular Organelles
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What reagents would you recommend for measuring the pH in acidic organelles, such as lysosomes?
For measuring the pH in acidic organelles, such as lysosomes, we recommend our LysoSensor ratio metric probes. Please see the links to the product page and descriptions below:
LysoSensor Yellow/Blue dextran, 10,000 MW, Anionic, Fixable (Cat. No. L22460):
LysoSensor Yellow/Blue dextran is a ratiometric probe that can be used to measure the pH of acidic organelles, such as lysosomes. This dextran conjugate is taken up by the cells through endocytosis. The LysoSensor dye produces blue fluorescence in neutral environments, but changes to yellow fluorescence in more acidic environments. The pKa of LysoSensor Yellow/Blue dextran is ~3.9.
LysoSensor Yellow/Blue DND-160 - Special Packaging (Cat. No. L7545):
LysoSensor Yellow/Blue DND-160 is a ratiometric probe that can be used to measure the pH of acidic organelles, such as lysosomes. The LysoSensor dye produces blue fluorescence in neutral environments, but changes to yellow fluorescence in more acidic environments. The pKa of LysoSensor Yellow/Blue DND-160 is ~4.2.
LysoSensor Blue DND-167 - Special Packaging (Cat. No. L7533):
LysoSensor Blue DND-167 can be used to measure the pH of acidic organelles, such as lysosomes. The LysoSensor dye becomes more fluorescent in acidic environments. The pKa of LysoSensor Blue DND-167 is ~5.1.
LysoSensor Green DND-189 - Special Packaging (Cat. No. L7535):
LysoSensor Green DND-189 can be used to measure the pH of acidic organelles, such as lysosomes. The LysoSensor dye becomes more fluorescent in acidic environments. The pKa of LysoSensor Green DND-189 is ~5.2.
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What is the difference between LysoSensor Yellow/Blue dextran, 10,000 MW, Anionic, Fixable (Cat. No. L22460) and LysoSensor Yellow/Blue DND-160 - Special Packaging (Cat. No. L7545)?
LysoSensor Yellow/Blue dextran, 10,000 MW, Anionic, Fixable (Cat. No. L22460) has an emission of 335/452, is fixable and allows loading of cells by endocytosis. The pKa of LysoSensor Yellow/Blue dextran is ~3.9, which is slightly lower than LysoSensor Yellow/Blue DND-160 - Special Packaging (Cat. No. L7545).
LysoSensor Yellow/Blue DND-160 - Special Packaging (Cat. No. L7545) has an emission of 329/440 and is unique as it shows both dual-excitation and dual-emission spectral peaks that are pH-dependent. Nevertheless, this LysoSensor only exhibits the pH-dependent dual-emission spectra in living cells. Lysosensor Yellow/Blue DND-160 has predominantly yellow fluorescence, and in less acidic organelles is has blue fluorescence. Dual-emission measurements may permit ratio imaging of the pH in acidic organelles such as lysosome.
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What is the recommended incubation time for LysoTracker Green DND-26 (Cat. No. L7526) and LysoSensor Yellow/Blue DND-160 (Cat. No. L7545)?
LysoTracker Green DND-26 (Cat. No. L7526) and LysoSensor Yellow/Blue DND-160 (Cat. No. L7545) show faster uptake in cells and consequently a faster "alkalizing effect" on the lysosomes, so we recommend incubating the cells with these probes for 1-5 min at 37 degrees C. As the other LysoTracker and LysoSensor probes have a slower rate of uptake, we recommend a longer incubation time of 30 min to 2 hr.
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