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What are the different cytotoxicity assays used for in-vitro toxicology?

The drug development industry has undertaken significant efforts to identify toxic events at the earliest opportunity during the development process. It has moved from a predominantly observational science at the level of disease-specific models to a more predictive model focused on target-specific, mechanism-based biological observations.

Our panel of fluorescence-based live cell assays are designed to help assess the impact of toxic agent on overall cell function. Our assays and probes have been developed to produce dyes that are sensitive, specific, and convenient. They offer increased photo stability, eliminate non-specific dye-associated artifacts to reduce false positives, and are compatible with common dyes and fluorescent markers for multiplex analysis. Our dyes are optimized for the most demanding imaging applications, including microplate-based high content screening, flow cytometry, confocal microscopy, and wide-field fluorescence microscopy where consistency and reproducibility are essential.

Our CELLESTIAL® assays are able to provide you with the ability to visualize toxic events in vitro. Our LYSO-ID® red cytotoxicity kits, green detection kits, and red detection kits are all members of the CELLESTIAL® product line, which includes reagents and assay kits comprising fluorescent molecular probes that have been extensively benchmarked for live cell analysis applications. CELLESTIAL® reagents and kits are optimal for use in demanding cell analysis applications involving confocal microscopy, flow cytometry, microplate readers and HCS/HTS.

Figure 1. 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 (ENZ-51015). Nuclei were counter-stained with Hoechst 33342 dye.

Figure 2. 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 U2-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.

Figure 3. LYSO-ID® Green dye (ENZ-51034) is a cell-permeant fluorescent probe that selectively associates with lysosomes and other acidic organelles (A). Cells pre-treated for 20 hours with weakly basic cell-permeant compounds, such as chloroquine, show dramatic increases in lysosome-like vesicle number and volume (B). Nuclei are counter-stained with Hoechst 33342 in the images.

Enzo Life Sciences’ ROS-ID® Total ROS/Superoxide detection kit (ENZ-51010) is designed to directly monitor real time reactive oxygen species (ROS) production in live cells using fluorescence microscopy or flow cytometry. The kit includes two fluorescent dyes as major components: Oxidative Stress Detection Reagent (Green) for total ROS detection reagent and Superoxide Detection Reagent (Orange). Through the combination of these two specific fluorescent probes, the kit provides a simple and specific assay for the real-time measurement of global levels of reactive oxygen species (ROS), and specifically, superoxide in living cells.

Figure 4. Profiling of reactive oxygen species formation by fluorescence microscopy was achieved in HeLa cells loaded with ROS/Superoxide detection reagents and treated with pyocyanin. General oxidative stress levels were monitored in the green channel, while superoxide production was detected in the orange channel. Pretreatment with NAC, a general ROS inhibitor, prevents formation of ROS (ENZ-51010).

Our NUCLEAR-ID® Green Chromatin Condensation Detection Kit detects nuclear condensation using a DNA intercalating dye that brightly stains the condensed chromatin of apoptotic cells, but only dimly stains the uncondensed chromatin of healthy, living cells. The cell-permeable dye used in this application is an aromatic, planar cationic structure that inserts between stacked base pairs on the DNA duplex, providing an environmentally-dependent fluorescence enhancement of the dye molecules and large increases in fluorescence signal relative to the free dye in solution. Since the signal enhancement provides a proportional response, direct quantitation of DNA is possible. Further signal increase is observed upon DNA condensation during apoptosis. Considering the general mutagenic effect of nucleic acid-binding dyes, careful storage and handling of this dye is recommended. A control apoptosis-inducing agent, staurosporine, is provided for monitoring apoptotic changes in nuclear organization. Potential applications for live-cell studies using the kit include monitoring the stages of chromatin condensation and rapid testing of compounds that induce apoptosis.

Figure 5. Flow cytometry analysis of chromatin condensation in response to application of environmentally toxic compounds. Analysis was performed following 16-hour treatment of 1 x 106 cells/ml Jurkat cells with various compounds at the indicated concentration. The 488nm excitable green-emitting dye eliminates the need for specialized UV lasers required by Hoechst dye-based assays (ENZ-51021).

The EFLUXX-ID® Multidrug Resistance Assay Kits allows for functional detection of all three clinically relevant ABC transporter proteins: MDR1 (p-glycoprotein), MRP1/2, and BCRP. The assay uses a hydrophobic, non-fluorescent compound that readily penetrates the cell membrane, where it is hydrolyzed to a hydrophilic fluorescent dye by intracellular esterases. Unless the EFLUXX-ID® dye is pumped out of the cell, the esterase cleaved dye is trapped inside the cell. Thus, cells exhibiting drug resistance will have diminished fluorescence. EFLUXX-ID® assay is the only available kit for the simultaneous monitoring of all three major ABC reporter proteins with the ability to profile individual pump activity. The proprietary AM-ester form of the EFLUXX-ID® dye is a hydrophobic non-fluorescent compound that readily penetrates the cell membrane and is subsequently hydrolyzed inside of the cells by intracellular esterases. The fluorescence signal of the dye generated within the cells depends upon the activity of the ABC transporters. The cells with highly active transporters will demonstrate lower fluorescence because of the active efflux of the probe from the cell. Application of specific inhibitors of the various ABC transporter proteins allows differentiation between the three common types of pumps.

Figure 6. Profiling of ABC transporter activity by known inhibitors was assessed in CHO K1 cells using eFluxx-ID Green and eFluxx-ID Gold dyes. Cells were incubated for 5 min at 37°C with general MDR Inhibitor (far left column) or transporter-specific inhibitors included in the kit. Cells were then loaded with the indicated dye for 30 min at 37°C and immediately analyzed by flow cytometry. Inhibitors used: 5 µM Cyclosporin A (general MDR inhibitor), 20 µM Verapamil (specific P-gp inhibitor), 0.05 mM MK-571 (specific MRP inhibitor), 0.05 mM Novobiocin (specific BCRP inhibitor).

Our long-standing flagship SCREEN-WELL® Compound Library portfolio offers an easy, ready-to-use method to streamline compound screening. Enzo offers focused compound libraries for organ-specific toxicology, as well as FDA approved drugs, natural products, chemical genomics, pathway targeting, and more. Our in-house chemical production capabilities, combined with our supplier network built over a 40 year history, gives us the ability to rapidly and inexpensively source traditional, custom, and bulk compounds in the gram to kilogram scale. We complete our sourcing using stringent quality control standards with state-of-the-art methods. SCREEN-WELL® libraries for nephrotoxicity, cardiotoxicity, hepatotoxicity, and hematopoietic toxicity contain compounds with defined and diverse organ-associated toxicity profiles. A variety of structurally and mechanistically different compound classes are included, as well as non-toxic controls. Compounds are dissolved in DMSO at 10mM and aliquoted into deep well plates at 100 or 500μl per well. The libraries are useful for predictive toxicology screening, including high-content protocols. You can better understand mechanisms of toxicity with your small molecule or candidate-drug of interest and compare your compound with known references for investigative toxicity while optimizing formulation efficacy and safety.

Growth in such early safety assessment initiatives has driven the need for more reliable and cost-effective high throughput in vitro toxicity assays capable of predicting toxic liabilities prior to investment in more costly pre-clinical and clinical trials. Enzo Life Sciences offers a range of products for your Toxicology research needs. With our selection of exceptional tools to identify phospholipidosis/steatosis such as our collection of LYSO-ID® kits, we are able to offer our end user a timely way to selectively stain the lysosome, quickly and effectively. Our SCREEN-WELL® Compound libraries offer the ability to work with a focused collection of compounds that are well defined and diverse for all of your predictive toxicology screening and assay development needs. As Scientists Enabling Scientists, we realize the value in providing relevant information to our customers working in the fields of life sciences, drug development and clinical research. We are happy to provide simple yet useful tips and guidance for your research needs. Please check out our successful research tips or contact our Technical Support Team for further assistance.

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