Genetic Engineering & Biotechnology News

OCT15 2017

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Genetic Engineering & Biotechnology News | GENengnews.com | OCTOBER 15, 2017 | 13 tual fate of apoptosis with in vitro systems) or in cases of other forms of necrosis that cause gross loss of membrane integrity, PS-driven Annexin V-NanoBiT complementation can occur either on the external or internal mem- brane surface. Figure 1 illustrates three stages of cells in the apoptotic process: healthy, early apoptosis, and secondary necrosis. In the new homogeneous method, a 2X reagent is added in the cell-culture medium of choice using the Annexin V-NanoBiT protein fusions, a luciferase substrate, and an optional pro-fluorescent cytotoxicity probe. Addition- al calcium chloride is provided to facilitate a maximized PS-annexin binding interaction. Reagent can be added directly to cell cultures at the same time as test compound is added. Figure 2 demonstrates an apoptotic re- sponse, initiated by an on-market antibody- drug conjugate, trastuzumab emtansine, ulti- mately resulting in secondary necrosis. Figure 3 illustrates a 48-hour kinetic trace of PS exposure and necrosis signal following treat- ment with the same drug. Real-Time Biomarker Detection during Extended Exposures Our improved, real-time annexin-based method is technically possible due to the novel chemical attributes of the luminogenic substrate and fluorogenic cytotoxicity probe. An esterified form of the luciferase substrate is delivered in great molar excess as part of the assay reagent, and becomes an active form of the substrate as it is de-protected. Esterase ac- tivity is inherent in viable cells as well as some serum supplements, and substrate de-protec- tion progresses at a steady and sustained rate in viable cells. In most instances, the active substrate is made available even during incu- bations up to 48 hours in length. An optional, unique fluorogenic cytotox- icity probe with affinity for DNA serves as a measure for loss of membrane integrity due to necrosis. The viable cell-impermeant probe produces nominal fluorescence signal in the absence of necrotic cells, but when cells lose membrane integrity, it complexes with ex- posed DNA, resulting in a substantial increase in fluorescence. This real-time necrosis probe in the reagent allows sequential measure of luminescence (as a result of PS exposure) and of fluorescence (as a result of necrosis) in the same sample to monitor cytotoxic progression. Use Time to Your Advantage Monitoring real-time PS exposure and ne- crosis during the induction of cell death may provide important contextual clues relating to mechanisms of action. For instance, strong lu- minescence signals resulting from PS exposure without measurable increases in fluorescence indicates classical early apoptotic phenotype. Likewise, strong luminescence signals with modest fluorescence increases may indicate a later maturing, dose-dependent apoptotic re- sponse. Conversely, strong and concurrent in- creases in both luminescence and fluorescence immediately following a stimulus would be consistent with primary necrosis. Therefore, relative kinetic emergence of these two bio- markers can help profile putative actions of a compound or biologic. One can multiplex RealTime-Glo Annexin V Apoptosis and Necrosis Detection Assay with several value-added endpoint chemistries to gather additional information regarding mechanisms of cell death. For instance, multi- plexing with cell viability reagents (e.g., Cell- Titer-Glo ® or CellTiter-Fluor™) can either pro- vide inversely correlated measures of cytotoxic- ity (by loss of signal) or reveal hidden cytostatic effects mediated by test agents. Similarly, cas- pase activity reagents (e.g., Caspase-Glo ® ) can provide orthogonal proof of the apoptotic phe- notype when counter-confirmation is required. Collection of these multiplexed signals is pos- sible due to either spectral compatibility of the individual signals or quenching attributes asso- ciated with reagent formulations. Conclusion The real-time, bioluminescent assay meth- od for apoptosis and necrosis described here provides full functionality of conventional (endpoint) annexin methods while requir- ing substantially less effort and being plate- based friendly. Time-resolved nature of the assay allows for dose-dependent character- ization of cytotoxic effects while providing kinetic resolution of cell death mechanisms of action. Finally, multiplexing the assay with other endpoint cell health assays can provide additional complementary or or- thogonal data. See more. Do more. • Maximize epitope diversity • Identify unique epitopes • Build your IP portfolio The Carterra™ LSA is a fully integrated antibody characterization platform that uses Array SPRi to analyze up to 384 binding interactions simultaneously, delivering 10x the data in 10% of the time with 1% of the sample require- ments of other systems. Combined with our application-focused analytical software, the LSA facilitates: • Epitope binning • Kinetic screening • Epitope mapping • Quantitation Contact us at: www.carterra-bio.com/lsa info@carterra-bio.com Toll free: 844-642-7635 Characterize the epitope landscape of your entire antibody library at the earliest stages of discovery to make smart decisions: Drug Discovery Tutorial Andrew Niles (andrew.niles@promega.com) is senior research scientist and Kevin Kupcho serves as a research scientist at Promega. Website: www.promega.com.

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