Lysosomal pH Activity in FaDu and HGFb Cells
A 4-channel confocal acquisition of FaDu and HGFb cells treated with autophagy modulators. Autophagosomes carry a tandem TagRFP/Emerald-GFP label (the GFP quenches below pH 6 while TagRFP stays stable) and lysosomes are marked with LysoTracker, giving a ratiometric readout of acidity. Cytely segments lysosomes and reports lysosome count per cell across drug-treatment groups including Bafilomycin and BEZ.
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Methodology
4-channel confocal Z-stacks. Ch1: TagRFP (acid-insensitive); Ch2: transmitted light; Ch3: Emerald GFP (acid-sensitive, quenches below pH 6); Ch4: LysoTracker (acid-sensitive, marks acidic lysosomes). Comparing the acid-sensitive and acid-insensitive channels gives a ratiometric readout of lysosomal/autolysosomal acidity.
Attribution
Balvan, Jan; Navratil, Jiri (2022). Autophagosomal/autolysosomal/lysosomal dynamics of FaDu and HGFb cells affected by autophagy modulators (dataset of dual labeling of autophagosomes and labeling lysosomes, confocal microscopy) [Data set]. Zenodo. https://doi.org/10.5281/zenodo.7116549 License: CC BY 4.0. Relevant paper: Hanelova K, Raudenska M, Kratochvilova M, Navratil J, Vicar T, Bugajova M, Gumulec J, Masarik M, Balvan J. Autophagy modulators influence the content of important signalling molecules in PS-positive extracellular vesicles. Cell Communication and Signaling. 2023;21(1):120. doi: 10.1186/s12964-023-01126-z.
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