Assay includes unique drug-like dye that rapidly partitions into cells and labels acidic organelles
Only commercial assay available that allows for long-term cell monitoring of cytotoxic effects
Multi-well, high-throughput with rapid 10-15 minute dye incubation
No co-incubation with artificial phospholipid analogs required for detection, eliminating the potential for confounding dye-associated artifacts
Monitors lysosome accumulation as a response to prolonged drug treatment
Quantitative results in as little as 3 hours, including drug treatment
The LYSO-ID® Red Cytotoxicity Kit monitors dysfunction of lysosomal degradation using a drug-like cationic amphiphilic tracer (CAT) dye that rapidly and selectively stains acidic organelles, and is suitable for monitoring accumulation of lysosomes and lysosome-like structures in live cells.
Mechanism of Action A cationic amphiphilic tracer (CAT) dye that rapidly partitions into cells in a similar manner as drugs that induce phospholipidosis. This tracer was created by placing titratable groups on the probe (dye), to enable labeling to be expanded into lamellar inclusion bodies of cells pretreated with weakly basic cell-permeant compounds, such as the antimalarial drug chloroquine. Besides lysosomes themselves, the probe can be employed for highlighting lysosome-like organelles under conditions wherein cells produce vacuoles that contain most of the degradative enzymes of the lysosome but are not as acidic as the parent organelle.
Eliminate Confounding Dye-associated Artifacts. The short 15-minute LYSO-ID® Red dye incubation eliminates the potential for confounding dye-associated artifacts. Relative fluorescent intensity of U-2 OS cells treated with chloroquine at different concentrations for 24 h. Cells stained with LipidTox dye (green line) were incubated in the presence of the fluorescent lipid for 24 h during treatment with the drugs. Cells stained with LYSO-ID® Red dye (red line) or Hoechst 33342 (blue line) were stained for 15 min after drug incubation.
High Throughput Screening of Therapeutics for Lysosome-perturbing Activity. Toxicity of Verapamil in U-2 OS cells was estimated using a conventional fluorescence microplate reader. U-2 OS cells were treated with Verapamil for 18 hours and stained with LYSO-ID® Red dye for 15 minutes. The high Z-factor (0.87 for 100 µM Verapamil) indicates that LYSO-ID® Red dye is suitable for HTS applications. Hoechst is used as a counterstain as a normalization control for cell number.
Composite bright-field and fluorescence microscopy images of control U2-OS cells (left) and cells pre-treated with 64µM Chloroquine for 5 hours (right). Cells were stained with LYSO-ID® Red dye for 10 minutes. Nuclei were counter-stained with Hoechst 33342 dye.
Excitation and emission spectra for A) LYSO-ID® Red Detection Reagent and B) Hoechst 33342 Nuclear Stain.
Drug-induced lysosome accumulation in U-2 OS cells was evaluated using LYSO-ID® Red dye. (A) Untreated cells (B) Chlorpromazine, 28 µM (C) Verapamil, 200 µM. These two compounds are cationic and amphiphilic, and known to induce abnormal accumulation of phospholipids within lysosomes, resulting in lamellar bodies. Treatment of U-2 OS cells with phospholipidosis-inducing drugs causes an increase in lysosome number and volume, detected as an increase in red fluorescence. Nuclei are counter-stained with Hoechst 33342 dye (blue).
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Product Details
Applications:
Fluorescence microscopy, Fluorescent detection
Application Notes:
96-well cell-based assay. Provides a rapid and quantitative approach for determining drug- or toxic agent-induced lysosome and lysosome-like organelle perturbations in live cells.
Quality Control:
A sample from each lot of LYSO-ID® Red cytotoxicity kit (GFP-CERTIFIED®) is used to stain epithelial cell line, MDCK, using the procedures described in the user manual. The Z’ value, as observed by the fluorescence microplate reader, is > 0.5 for cells treated with 120µM verapamil. The blue nuclear counterstain is at least 70% of the value for the untreated cells.
Quantity:
2 x 96-well plates
Use/Stability:
With proper storage, the kit components are stable up to the date noted on the product label. Store kit at -20°C in a non-frost free freezer, or -80°C for longer term storage.
Handling:
Protect from light. Avoid freeze/thaw cycles.
Shipping:
Shipped on Dry Ice
Long Term Storage:
-80°C
Contents:
10x dual color detection reagent: 2 x 1ml Detection buffer: 20ml Verapamil control: 3μmoles 10x assay buffer: 15ml
Regulatory Status:
RUO - Research Use Only
Product Literature References
A cell-based assay using HepaRG cells for predicting drug-induced phospholipidosis: T. Tomida, et al.; J. Toxicol. Sci. 42, 641 (2017), Application(s): Microplate reader measurements using HepaRG cells, Abstract;
SerpinB2 (PAI-2) modulates proteostasis via binding misfolded proteins and promotion of cytoprotective inclusion formation: J.A. Lee, et al.; PLoS One 10, e0130136 (2015), Application(s): Autophagy response measured by fluorescence microplate reader, Abstract; Full Text
Involvement of interleukin-1β in the autophagic process of microglia: relevance to Alzheimer's disease: A. François, et al.; J. Neuroinflammation 10, 151 (2013), Application(s): Measurement with a microplate reader, Abstract; Full Text
The full-of-bacteria gene is required for phagosome maturation during immune defense in Drosophila: M.A. Akbar, et al.; J. Cell Biol. 192, 383 (2011), Abstract;
A live-cell fluorescence microplate assay suitable for monitoring vacuolation arising from drug or toxic agent treatment: J. Coleman, et al.; J. Biomol. Screen. 15, 398 (2010), Abstract;
Automated detection of drug-induced lysosomal cytotoxicity - automation of the Lyso-ID Red assay using the EL406 combination washer dispenser: P. Held, et al.; Lab Manager Magazine June, (2010), Full Text
General Literature References
Intense pseudotransport of a cationic drug mediated by vacuolar ATPase: procainamide-induced autophagic cell vacuolization: G. Morissette, et al.; Toxicol. Appl. Pharmacol. 228, 364 (2008), Abstract;
Drug-induced phospholipidosis: N. Anderson & J. Borlak; FEBS Lett. 580, 5533 (2006), Abstract;
N-substituted 4-aminobenzamides (procainamide analogs): an assessment of multiple cellular effects concerning ion trapping: G. Morissette, et al.; Mol. Pharmacol. 68, 1576 (2005), Abstract;
Massive cell vacuolization induced by organic amines such as procainamide: G. Morissette, et al.; J. Pharmacol. Exp. Ther. 310, 395 (2004), Abstract;
Quantitation of the lysosomotropic character of cationic amphiphilic drugs using the fluorescent basic amine Red DND-99: B.Lemieux, et al.; Anal. Biochem. 327, 247 (2004), Abstract;
