PROTACs is an emerging strategy for targeted therapy in drug discovery. After target protein degradation, PROTAC is released and continues the degradation process. Thus, toxicological issue is one of the main challenges for PROTACs application in drug delivery. Cell cytotoxicity assay is a common method in investigation of the toxicity of molecules on cellular systems, Profacgen offers multiple assays to help our clients address this issue in vitro.
Enzyme leakage assays
The detection of released enzymes is used to quantitate the leakage of intracellular proteins after membrane disintegration, which is resulted from cell membrane damage. Contrary to traditional radiological methods, the increasing diversity of fluorescents makes it convenient and secure to detection.
Membrane impermeable dyes
Membrane impermeable fluorescent dyes is widely adopted for damaged cell membranes in studying cellular metabolism and viability assays. This kind of molecules are excluded from cellular membrane, but transports freely through the permeabilized membranes of dead and dying cells, which is captured by a flurescent detection system.
Amine-reactive dyes are a kind of viability dyes for identifying dead cells. The dyes cross cell membranes of dead cells and irreversibly react with free amines in cytoplasm. In live cells, these dyes are excluded from the intact cell membranes, and free dye is washed away after staining. Since amine-reactive dyes are fluorescent when excited by lasers, dead cells could be identified by flow cytometry.
Dye combination assays
This method utilizes multiple dyes combined in a sing live-dead cell assay, which is possible to quantify cell death induced by a variety of different agonists or label dead and live cells concurrently. Moreover, using a single known cytotoxic agent as a positive control for multivariate classifier allowed accurate quantification of cytotoxicity enabling generation of dose–response curves.
Apoptosis is a form of cell death characterized by several features including cell shrinkage, membrane blebbing, chromosome condensation, nuclear fragmentation. Apoptotic cells are recognized on the basis of reduced DNA content and morphological changes that include nuclear condensation, which can be detected by flow cytometry or different dyes.
Cell proliferation and cell cycle assays
Cell proliferation is an increase in cell number in tissue or culture plates. Cells proliferate by a process known as Cell Cycle in optimal conditions. Cell cycle is a cellular event where one cell divides itself into two daughter cells. Once any process goes wrong, cell cycle halts duly, activates pathway to repair the damage, or activates the apoptotic machinery. Cell proliferation and cell cycle can be monitored by measuring metabolic activity like MTT assay, XTT assay, WST-1 assay, luciferase assay and so on.
Cell viability assays
Cell viability assay is widely used for screening compounds, measuring receptor binding and signal transduction events. The purpose of cell viability assays is to determine the number of living cells in a population by a variety of methods including cellular metabolism and enzyme activity, factors detection, flow cytometry and imaging technique.
Cell-based assays are commonly used in measuring the results of cell proliferation, testing for cytotoxic effects, and determining viable cell number. As an experienced service provider, Profacgen owns complete experimental platform and professional scientist team.
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2. Perfetto SP, Chattopadhyay PK, Lamoreaux L, Nguyen R, Ambrozak D, Koup RA, Roederer M. (2010). Amine-Reactive Dyes for Dead Cell Discrimination in Fixed Samples. Current Protocols in Cytometry, 53(1), 9.34.1–9.34.14.doi:10.1002/0471142956.cy0934s53
3. Oancea M, Mazumder S, Crosby ME, Almasan A. (2006.). Apoptosis Assays. Methods in Molecular Medicine, 129:279-90. doi: 10.1385/1-59745-213-0:279