Frequently Asked Questions
Caspase-Glo® 3/7 Assay System
The complete protocol for this product is available in Technical Bulletin #TB323.
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 relative luminescence output will vary with different instruments, this variation should not affect assay performance.
What experimental controls should I use?
- Blank (background) reaction: Caspase-Glo® 3/7 Reagent, vehicle and cell culture medium without cells.
- Negative control: Caspase-Glo® 3/7 Reagent and vehicle-treated cells in medium.
- Positive control: Caspase-Glo® 3/7 Reagent and cells treated with a known apoptosis inducer with your sample type under your test conditions for comparison.
What are the main assay considerations?
- It is important that cells are as healthy as possible before testing. Handle cells gently and follow culturing recommendations for the specific cell type.
- Apoptosis is a dynamic process, so it is important to optimize the dose, timing and cell number at which the maximum activity of caspase 3/7 can be detected.
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