RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay

Protocol tips

Upstream tips	Protocol tips	Downstream tips
• No-wash, one-step Annexin V assay • Non-lytic. Allows continual monitoring of cell state to determine apoptotic onset • Scalable for high-throughput screening applications	• Seed 10,000 cells in 100μL/well on 96 white flat-bottomed multi-wells, and incubate overnight at 37◦C to allow adhesion.

Upstream tips
• No-wash, one-step Annexin V assay • Non-lytic. Allows continual monitoring of cell state to determine apoptotic onset • Scalable for high-throughput screening applications
Protocol tips
• Seed 10,000 cells in 100μL/well on 96 white flat-bottomed multi-wells, and incubate overnight at 37◦C to allow adhesion.

Publication protocol

The RealTime-Glo™Annexin V Apoptosis and Necrosis Assay kit (Promega) was used following the manufacturer’s protocol. For both HepG2 and A549, 10,000 cells in100μL/well were seeded on 96 white flat-bottomed multi-wells and incubated overnight at 37◦C to allow adhesion. After 24 h, the medium was removed and cells were treated with the three highest concentrations of MZ (at 295.7, 29.6 and 2.96μM) or ZOX (at 463.4,46.34 and 4.63μM), or vehicle as control (DMSO 0.8% and 0.2%) in duplicated wells, and each being concentrated two-fold. Then, 100μL/well of reagent mix solution was added to reach nominal concentrations. Plates were incubated at 37◦C and they were read each hour for the first 8 h and then at 24 h for both luminescence and fluorescence (485/535 nm)by a Victor 3 Multilabel Reader (PerkinElmer), detecting apoptosis and necrosis signals, respectively

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Manufacturer protocol

Download the product protocol from Promega for RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay below.

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