Apoptosis Frequently Asked Questions

• Can I quantify apoptosis in any cell type?
• Can I use non-adherent cells?
• Can I use cells pre-labelled with a fluorescent probe?
• Can I multiplex several markers of apoptosis together?
• Can I multiplex apoptosis with other cell health readouts, such as cytotoxicity?
• Can I multiplex apoptosis with Propidium Iodide?
• Can I use the IncuCyte® Apoptosis Reagents on an IncuCyte® FLR?
• What IncuCyte® objective should I use for quantifying apoptosis?
• Should I use a specific micro-titre plates for measuring apoptosis?
• Do I need to wash or fix cells after adding the IncuCyte® Apoptosis Reagents?
• Can I use my standard cell culture media for the duration of the assay?
• Can I perform cell counting when using the IncuCyte® Apoptosis Reagents?
• Can I normalise apoptosis measurements to a measurement of cell proliferation?

Can I quantify apoptosis in any cell type?

Yes, in principle. The Caspase-3/7 and Annexin V protocols have been optimised using a range of adherent and non-adherent cell lines and should therefore be applicable to any cell type including cancer cells, immune cells and neurons. Please note some cell types may lack expression of Caspase-3 (e.g. MCF-7 cells). For these cells types we would recommend using the IncuCyte® Annexin V Reagents rather than Caspase-3/7 Reagent for apoptosis measurements.

Can I use non-adherent cells?

Yes. The protocols and data described here show that it is possible to quantify apoptosis in suspension cell lines such as Jurkat human T lymphocyte cells. You may want to coat your plate to ensure your suspension cells stay in the field of view. Coatings such as poly-L-ornithine, poly-D-lysine or Matrigel™ have been shown to work well.

Can I use cells pre-labelled with a fluorescent probe?

Yes. As long as the fluorophore is compatible with the IncuCyte® Live-Cell Analysis System and does not interfere with the fluorescence emitted by the IncuCyte® Caspase3/7 or  Annexin V Reagents used.

Can I multiplex several markers of apoptosis together?

Yes. The protocols and data described here show that it is possible to quantify two markers of apoptosis (e.g. caspase-3/7 activation and phosphatidylserine externalization) in a single well. For this we recommend that you duplex IncuCyte® Annexin V Red Reagent (Cat No. 4641) with the green IncuCyte® Caspase-3/7 Reagent (Cat No. 4440).

Can I multiplex apoptosis with other cell health readouts, such as cytotoxicity?

Yes. Duplex the IncuCyte® Caspase-3/7 Reagent (Cat No. 4440) with the IncuCyte® Red Cytotox Reagent (Cat No. 4633) or combine the opposing red and green versions of the IncuCyte® Annexin V Reagent (Cat No. 4641 or 4642) with IncuCyte® Cytotox Reagents (Cat No. 4632 or 4633).

Can I multiplex apoptosis with Propidium Iodide?

Yes. It is possible to duplex the IncuCyte® Annexin V Red Reagent (Cat No. 4641) with Propidium Iodide. We recommend quantifying the Propidium Iodide signal using the red channel of the IncuCyte® system and setting a spectral unmixing parameter of 80% Red from Green (please refer to the Fluorescent Dye Optimization Technical Note for more information).

Can I use the IncuCyte® Apoptosis Reagents on an IncuCyte® FLR?

Yes. Both the IncuCyte® Caspase-3/7 Reagent (Cat No. 4440) and IncuCyte® Green Annexin V Reagent (Cat No. 4642) are compatible with the IncuCyte® FLR system. Please note that the IncuCyte® Red Annexin V Reagent (Cat No. 4641) is not compatible with the excitation/emission spectra of the IncuCyte® FLR.

What IncuCyte® objective should I use for quantifying apoptosis?

Data has been generated with both the 10x and 20x objectives. The 10x objective offers the benefit of a greater field of view and reduces the number of images required per well. For higher spatial resolution the 20x objective is recommended.

Should I use a specific micro-titre plates for measuring apoptosis?

IncuCyte® Apoptosis Assays can be performed using any clear sided or black sided 96 or 384-well clear-bottomed micro-titre plate that is supported on the IncuCyte® ZOOM system. We have used Corning 96-well plates (Cat # 3904) for the experiments described here.

Do I need to wash or fix cells after adding the IncuCyte® Apoptosis Reagents?

No. The IncuCyte® Annexin V Reagents have been specially formulated for live-cell imaging with the IncuCyte® system. Simply mix-and-read. Washing or fixing is not required.

Can I use my standard cell culture media for the duration of the assay?

Yes. There is no requirement to use a specialised assay media or buffer when using IncuCyte® Annexin V or Caspase-3/7 Reagents with the IncuCyte® system. Integrated IncuCyte® image analysis tools enable you to minimize any background fluorescence that may arise from the media, reagents or test agents used. Please do note however that binding of Annexin V to externalised phosphatidylserine is Ca²⁺-dependent, therefore ensure the Ca²⁺ concentration in the assay media is ≥1 mM.

Can I perform cell counting when using the IncuCyte® Apoptosis Reagents?

We recommended counting apoptotic cells when using the IncuCyte® Caspase3/7 Reagent for Apoptosis. The Caspase-3/7 Reagent labels the nuclei of apoptotic cells so it is easy to differentiate between the discretely labelled nuclei of neighbouring apoptotic cells. The IncuCyte® Annexin V Reagents, however, label the entire cell surface. As such, distinguishing neighbouring cells can be challenging. When using Annexin V Reagents we recommend quantifying the total fluorescence area as this is a more robust approach.

Can I normalise apoptosis measurements to a measurement of cell proliferation?

Yes, measurements of apoptosis can be normalised to label-free IncuCyte® cell confluence measurements. Although some treatments may cause significant cell fragmentation or substantial changes in cell phase contrast we have found the IncuCyte® phase contrast confluence metric to be an informative estimate of total cell number over the course of the assay.

It is also possible to normalise to the total number of DNA containing objects at the end of the apoptosis assay. We recommend using Vybrant® DyeCycle™ Green Stain (ThermoFisher) for this purpose. The stain can be added directly to the assay wells without aspiration or washing of the apoptosis reagent containing media.