Lysosome and acidic-vesicle live-cell staining
Materials Required
Principle
Lysosome and acidic-vesicle live-cell staining detects acidic intracellular compartments by using membrane-permeant acidotropic probes that accumulate in low-pH vesicles, including lysosomes, late endosomes, autolysosomes, and acidic phagosomes[1][2][3][4]. LysoTracker staining is commonly used as an intensity-based readout of acidic lysosomal compartment abundance or enlargement, while acridine orange produces green fluorescence in less concentrated compartments and red fluorescence after concentration-dependent accumulation in acidic vesicular organelles[2][3]. Loss or reduction of acridine-orange red signal can be used as a readout of lysosomal membrane permeabilization or reduced acidic-vesicle integrity[3][4]. This protocol is designed for live cultured cells and can be adapted for fluorescence microscopy, high-content imaging, plate-reader readout, or flow cytometry when the selected literature supports the readout[1][2][3][4]. Because these dyes report acidotropic accumulation rather than lysosome identity alone, interpretation should be cross-checked with lysosomal markers or complementary assays when lysosome specificity is required[1][3][5].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Use live-cell-compatible culture medium for dye dilution and imaging, phosphate-buffered saline for washing when washing is included in the published procedure, and acridine orange when acidic-vesicle volume or lysosomal membrane permeabilization is the intended readout[3][4].
• Bafilomycin A1 may be used as a control for loss of vesicle acidification when the experiment specifically tests pH-dependent dye accumulation[3][4].
• LysoTracker Red DND-99 is used to label acidic lysosomal compartments in live cells and has been applied at 50 nM in patient-derived fibroblast lysosomal-storage-disease assays and at 500 nM in mouse embryo programmed-cell-death staining protocols[1][2][6].
• Acridine orange is used to label acidic vesicular organelles and to quantify red/green fluorescence ratios in microscopy, flow cytometry, or plate-reader formats[3][4].
• Cresyl violet is an alternative membrane-permeant fluorescent lysosomal marker that localizes to lysosomes and acidic vacuoles and was reported to be less photobleaching-prone than several alternative acidotropic probes[5].
• A fluorescence microscope, confocal microscope, high-content imager, fluorescence plate reader, or flow cytometer can be used depending on whether the endpoint is cellular localization, image-based intensity, red/green acridine-orange ratio, or population-level fluorescence[1][2][3][4].
• Live-cell imaging should use temperatureand atmosphere-compatible conditions when cells require physiological culture conditions during imaging[1][3].
• Bafilomycin A1 may be used as a control for loss of vesicle acidification when the experiment specifically tests pH-dependent dye accumulation[3][4].
• LysoTracker Red DND-99 is used to label acidic lysosomal compartments in live cells and has been applied at 50 nM in patient-derived fibroblast lysosomal-storage-disease assays and at 500 nM in mouse embryo programmed-cell-death staining protocols[1][2][6].
• Acridine orange is used to label acidic vesicular organelles and to quantify red/green fluorescence ratios in microscopy, flow cytometry, or plate-reader formats[3][4].
• Cresyl violet is an alternative membrane-permeant fluorescent lysosomal marker that localizes to lysosomes and acidic vacuoles and was reported to be less photobleaching-prone than several alternative acidotropic probes[5].
• A fluorescence microscope, confocal microscope, high-content imager, fluorescence plate reader, or flow cytometer can be used depending on whether the endpoint is cellular localization, image-based intensity, red/green acridine-orange ratio, or population-level fluorescence[1][2][3][4].
• Live-cell imaging should use temperatureand atmosphere-compatible conditions when cells require physiological culture conditions during imaging[1][3].
Experimental Procedure
• Seed live adherent cells in optically compatible culture plates or coverslip chambers at a density that allows single-cell or field-level fluorescence measurement without excessive overlap[1][2][3].
• Prepare dye working solutions freshly in cell culture medium; reported LysoTracker Red DND-99 concentrations include 50 nM for human fibroblast lysosomal-storage-disease assays and 500 nM for embryo and differentiating embryonic-stem-cell staining, while acridine orange protocols commonly analyze acidic vesicular organelles by measuring red and green fluorescence channels after live-cell staining[2][3][6].
• Include untreated cells as a baseline control and include a condition expected to reduce vesicle acidification or disrupt lysosomal integrity only when the biological question requires such validation[3][4].
• For LysoTracker staining, replace or supplement culture medium with LysoTracker-containing medium and incubate live samples using a literature-supported concentration selected for the sample type; 50 nM LysoTracker Red DND-99 was optimal in NPC patient-derived fibroblasts in the Xu et al. assay, while 500 nM LysoTracker was used in the JoVE embryo and differentiating embryonic-stem-cell protocol[2][6].
• After staining, acquire fluorescence images or plate-reader measurements under the same exposure and acquisition settings across experimental groups[1][2].
• For acridine-orange staining, stain live cells with acridine orange and acquire both green and red fluorescence channels because the red signal reflects concentration-dependent accumulation in acidic vesicular organelles and the red/green relationship is used for ratiometric analysis[3].
• For lysosomal membrane permeabilization assays, monitor the loss or redistribution of acridine-orange fluorescence over time using microscopy, flow cytometry, or a microplate reader, because published methods use acridine orange to follow lysosomal destabilization in live cells[4].
• For cresyl-violet staining, use it as an alternative acidic-compartment marker when compatibility with green, red, or far-red fluorescent probes is needed, because it was characterized as a membrane-permeant lysosomal and acidic-vacuole marker across yeast, Drosophila, human, murine, and canine cells[5].
• Do not interpret cresyl violet, LysoTracker, or acridine orange as exclusive lysosome markers without validation because acidotropic accumulation can include multiple acidic organelles[1][3][5].
• For LysoTracker assays, quantify fluorescence intensity per well, per cell, or per field, and compare each experimental condition with matched untreated or healthy-control cells because LysoTracker intensity was used to detect enlarged lysosomal compartments in patient-derived fibroblasts[2].
• For acridine orange, analyze red fluorescence, green fluorescence, or red/green ratio according to the chosen readout, because red acridine-orange fluorescence represents dye accumulation in acidic vesicular organelles while ratiometric analysis was developed to study acidic organelles and autophagy[3].
• Interpret reduced LysoTracker or acridine-orange acidic-vesicle signal cautiously because it may reflect decreased vesicle acidity, altered vesicle volume, altered dye uptake, phototoxicity, or membrane permeabilization depending on the assay context[3][4].
• Use identical acquisition settings across samples within one experiment and report the dye, concentration, incubation conditions, imaging platform, channel settings, and analysis unit because published protocols rely on these parameters for reproducible comparison[1][2][3][4].
• Prepare dye working solutions freshly in cell culture medium; reported LysoTracker Red DND-99 concentrations include 50 nM for human fibroblast lysosomal-storage-disease assays and 500 nM for embryo and differentiating embryonic-stem-cell staining, while acridine orange protocols commonly analyze acidic vesicular organelles by measuring red and green fluorescence channels after live-cell staining[2][3][6].
• Include untreated cells as a baseline control and include a condition expected to reduce vesicle acidification or disrupt lysosomal integrity only when the biological question requires such validation[3][4].
• For LysoTracker staining, replace or supplement culture medium with LysoTracker-containing medium and incubate live samples using a literature-supported concentration selected for the sample type; 50 nM LysoTracker Red DND-99 was optimal in NPC patient-derived fibroblasts in the Xu et al. assay, while 500 nM LysoTracker was used in the JoVE embryo and differentiating embryonic-stem-cell protocol[2][6].
• After staining, acquire fluorescence images or plate-reader measurements under the same exposure and acquisition settings across experimental groups[1][2].
• For acridine-orange staining, stain live cells with acridine orange and acquire both green and red fluorescence channels because the red signal reflects concentration-dependent accumulation in acidic vesicular organelles and the red/green relationship is used for ratiometric analysis[3].
• For lysosomal membrane permeabilization assays, monitor the loss or redistribution of acridine-orange fluorescence over time using microscopy, flow cytometry, or a microplate reader, because published methods use acridine orange to follow lysosomal destabilization in live cells[4].
• For cresyl-violet staining, use it as an alternative acidic-compartment marker when compatibility with green, red, or far-red fluorescent probes is needed, because it was characterized as a membrane-permeant lysosomal and acidic-vacuole marker across yeast, Drosophila, human, murine, and canine cells[5].
• Do not interpret cresyl violet, LysoTracker, or acridine orange as exclusive lysosome markers without validation because acidotropic accumulation can include multiple acidic organelles[1][3][5].
• For LysoTracker assays, quantify fluorescence intensity per well, per cell, or per field, and compare each experimental condition with matched untreated or healthy-control cells because LysoTracker intensity was used to detect enlarged lysosomal compartments in patient-derived fibroblasts[2].
• For acridine orange, analyze red fluorescence, green fluorescence, or red/green ratio according to the chosen readout, because red acridine-orange fluorescence represents dye accumulation in acidic vesicular organelles while ratiometric analysis was developed to study acidic organelles and autophagy[3].
• Interpret reduced LysoTracker or acridine-orange acidic-vesicle signal cautiously because it may reflect decreased vesicle acidity, altered vesicle volume, altered dye uptake, phototoxicity, or membrane permeabilization depending on the assay context[3][4].
• Use identical acquisition settings across samples within one experiment and report the dye, concentration, incubation conditions, imaging platform, channel settings, and analysis unit because published protocols rely on these parameters for reproducible comparison[1][2][3][4].
Troubleshooting
Weak LysoTracker signal:
The selected concentration may be below the useful range for the cell type or vesicle burden.Optimize within literature-supported examples rather than assuming one universal concentration, because 50 nM LysoTracker Red DND-99 was sufficient in NPC fibroblasts whereas 500 nM was used in embryo and differentiating embryonic-stem-cell staining[2][6].
Strong acridine-orange green signal but weak red acidic-vesicle signal:
Acidic-vesicle accumulation may be reduced, vesicle acidity may be impaired, or lysosomal membrane integrity may be compromised.Analyze red/green fluorescence and include an acidification-disruption or lysosomal-destabilization control only when experimentally justified, because acridine-orange red fluorescence depends on accumulation in acidic vesicular organelles and real-time acridine-orange assays monitor lysosomal membrane permeabilization[3][4].
Fluorescent puncta are interpreted as lysosomes without validation:
Acidotropic dyes can accumulate in acidic compartments beyond lysosomes.Describe the readout as acidic vesicles or acidic organelles unless lysosome identity is validated with a lysosomal marker or complementary assay[1][3][5].
Loss of signal during repeated imaging:
Some fluorescent lysosomal probes can be affected by photobleaching or photodamage during live imaging.Minimize repeated light exposure and consider cresyl violet when compatible, because cresyl violet was reported to localize to acidic compartments and to show reduced photobleaching relative to alternative acidotropic probes[5].
References:
- [1]. Dolman NJ, et al. A Review of Reagents for Fluorescence Microscopy of Cellular Compartments and Structures, Part I: BacMam Labeling and Reagents for Vesicular Structures. Curr Protoc. 2023;3(6):e751. [Content Brief]
- [2]. Xu M, et al. A phenotypic compound screening assay for lysosomal storage diseases. J Biomol Screen. 2014;19(1):168-175. [Content Brief]
- [3]. Thomé MP, et al. Ratiometric analysis of Acridine Orange staining in the study of acidic organelles and autophagy. J Cell Sci. 2016;129(24):4622-4632. [Content Brief]
- [4]. Eriksson I, et al. Real-Time Monitoring of Lysosomal Membrane Permeabilization Using Acridine Orange. Methods Protoc. 2023;6(4):72. [Content Brief]
- [5]. Ostrowski PP, et al. Cresyl violet: a superior fluorescent lysosomal marker. Traffic. 2016;17(12):1313-1321. [Content Brief]
- [6]. Fogel JL, et al. Use of LysoTracker to detect programmed cell death in embryos and differentiating embryonic stem cells. J Vis Exp. 2012;(68):4254. [Content Brief]
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