IncuCyte® Phagocytosis Assay
Frequently Asked Questions

• Can I quantify phagocytosis in any phagocytic cell type?
• Can I use non-adherent phagocytic cells?
• Can I use phagocytes pre-labelled with a fluorescent probe?
• Can I use other bioparticles conjugated to pHrodo® or another pH sensitive fluorophore?
• Can I multiplex phagocytosis with cell health readouts, such as cytotoxicity or apoptosis?
• Can I use the IncuCyte® pHrodo® Bioparticles® on an IncuCyte® FLR?
• What objective should I use for quantifying phagocytosis?
• Am I constrained to specific micro-titre plates for measuring phagocytosis?
• Do I need to fix cells after using pHrodo Bioparticles?
• Can I use my standard cell culture media for the duration of the assay?
• Can I normalise the data to cell number, thus removing the impact of phagocyte proliferation when measuring phagocytosis over long time periods?

Can I quantify phagocytosis in any phagocytic cell type?

Yes, in principle. Although the protocols and data described have been optimised using J774A.1 mouse macrophages the method should be applicable to any phagocytic cell type.

Can I use non-adherent phagocytic cells?

Yes. Whilst we have focussed on J774A.1 adherent cells in developing the phagocytosis application, the use of non-adherent phagocytic cell types should be possible. Care must be taken to ensure cells remain in the field of view - this may be facilitated by the use of plate coatings such as poly-D-lysine or Matrigel™.

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

Yes. As long as the fluorophore is compatible with IncuCyte® Live-Cell Analysis System and does not interfere with the fluorescence emitted by the IncuCyte® pHrodo® Bioparticles®.

Can I use other bioparticles conjugated to pHrodo® or another pH sensitive fluorophore?

Yes. As long as the fluorophore is compatible with the excitation/emission spectra for the IncuCyte® Live-Cell Analysis System.

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

Yes. We recommend that you duplex green fluorescent cytotoxicity or apoptosis reagents (e.g. IncuCyte® Kinetic Caspase-3/7 Apoptosis Assay Reagent (Cat No 4440) with IncuCyte® pHrodo® Red Bioparticles® to avoid spectral compatibility issues.

Can I use the IncuCyte® pHrodo® Bioparticles® on an IncuCyte® FLR?

Yes. However, please ensure you are using the IncuCyte® pHrodo® Green Bioparticles® as these are compatible with the excitation/emission spectra of the IncuCyte® FLR.

What objective should I use for quantifying phagocytosis?

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 (e.g. evidence of subcellular localisation) the 20x objective is recommended.

Am I constrained to specific micro-titre plates for measuring phagocytosis?

No. We have used Corning 96-well plates (Cat # 3595) for the experiments described here, however all microtitre plates supported on the IncuCyte® Live-Cell Analysis System should be compatible with the phagocytosis application. We recommend IncuCyte® ImageLock 96-well Plates (Cat No 4379) for creating high resolution movies.

Do I need to fix cells after using pHrodo Bioparticles?

No. The use of IncuCyte® pHrodo® Bioparticles with the IncuCyte® Live-Cell Analysis System is fully compatible with living cells. 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 buffer when using IncuCyte® pHrodo® Bioparticles with the IncuCyte® Live-Cell Analysis System. Background fluorescence arising from the media and the IncuCyte® pHrodo® Bioparticles per se can be miminised using the IncuCyte software analysis tools (see the Detailed Protocol).

Can I normalise the data to cell number, thus removing the impact of phagocyte proliferation when measuring phagocytosis over long time periods?

Yes, but you will need to count cells over time using fluorescent nuclear labelling for the normalisation. Attempts to measure cell confluence using the phase contrast algorithm may be compromised by the presence of the bioparticles.