Frequently Asked Questions
RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay
The complete protocol for this product is available in Technical Manual #TM507.
What type of multiwell plates should I use for this assay?
For optimum performance, use opaque, white multiwell plates that are compatible with your luminometer. Luminescence signal is diminished in black plates and increased well-to-well cross talk is observed in clear plates. RLU values, such as those shown in the figures of the Technical Bulletin, will vary depending on which plates and luminometers are used to generate data. Although relative luminescence output will vary with different instruments, this variation should not affect assay performance. Any solid white plates that fit your luminometer should be suitable. We don’t have specific recommendations but here are some examples:
- Corning® Costar plate (Cat.# 3917)
- Greiner Bio-One CELLSTAR plate (Cat.# 655073)
- Nunc™ MicroWell™ 96-Well, Nunclon Delta-Treated (Cat.# 136101/02)
- Falcon® 96-well Solid White Flat Bottom TC-treated (Cat.# 353296)
What instrument settings should I use for this assay?
Most standard plate readers designed for measuring luminescence are suitable for this assay. Some instruments do not require gain adjustment, while others may require optimization of the gain settings to achieve sensitivity and dynamic range. An integration time of 0.3–1 second per well should serve as guidance. For exact instrument settings, consult your instrument manual. Although raw relative luminescence output will vary with different instruments, this variation should not affect assay performance.
What time points should I take?
When testing the effects of a compound on cell health, the kinetics of cytotoxicity often differ depending on a wide variety of factors such as the cell line, compound type, compound concentration or stimulus intensity, and exposure time. Using a positive control with a time course between 2 to 72 hours will help identify the window of apoptosis for your experiment.
Are there any considerations when preparing the reagent?
We recommend including serum supplements, which greatly enhance solubility of the substrate and calcium; the annexin V-PS binding interaction is Ca2+-dependent.
What are the appropriate controls to include?
- Include a known apoptotic inducer for positive controls, no-compound (vehicle only) controls, no-cell background controls and digitonin for total cell lysis.
- The necrosis reagent is vital to help distinguish between apoptosis and secondary necrosis.
For additional information, view the product page.
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