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of when changes happen. Te assay measures translocation of phosphatidylserine (PS) from the inner to outer membrane leaflet that is a hallmark of healthy cells transitioning to apoptosis. It uses annexin-V-fusion proteins that contain binary subunits of a luminescent


enzyme (NanoBit), which are drawn into complementing proximity only due to their affinity for PS. In the presence of a time-released substrate, the complemented enzymes report real-time PS exposure. Te Necrosis Detection Reagent reports changes in membrane integrity as a result of necrosis.


Fig. 1. Real-time-Glo Annexin V Apoptosis Assay


Together, these real-time measures establish the mechanism of action (apoptosis, primary necrosis, or alternative programs) for cell death.


A dose response experiment was done with the proteasome inhibitor bortezomib and the leukemic cell line K562. Measurement of luminescence and fluorescence was done by the Clariostar every 15 minutes for 48 hours. Te experiment revealed the exposure of phosphatidylserine (luminescence) before the necrotic response (fluorescence) indicating the expected apoptotic mechanism for this therapeutic proteasome inhibitor. Te results show a combination of a novel assay methodology and instrumentation that allows the user to walk away and return to outstanding results. Te real-time nature of the results allows capturing information that would require extensive effort to be achieved using previous apoptosis assay techniques.


Fig. 2. Simultaneous measurement of PS positivity (purple) and membrane integrity (orange)


www.scientistlive.com


Andrea Krumm is with BMG Labtech. www.bmglabtech.com


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