Flow cytometry allows you to have the flexibility to evaluate samples from multiple species in a single experiment. The key to making this work is having flow cytometry staining panels that are cross-reactive and still allow discrete cells to be distinguished from each other in a single run.

Consider these points as you plan a multi-species flow cytometry assay:

1. Which species has fewer available reagents? If you are staining both mouse and human samples with specific markers, you may discover fewer antibodies are available to stain particular human biomarkers. In these situations when a species has fewer available reagents, you will have to build your staining panel around your reagent with the fewest antibody options.
2. Think about secondary antibodies. If you are using primary antibodies that are not conjugated with fluorescent dyes, you will need to stain them with a fluorescently labeled secondary antibody. But, when mixing species, avoid using the same secondary to stain the two different species. Your primary antibodies for both mouse and human cells might be made in rabbit, so you will need two different fluorescently-dyed anti-rabbit antibodies to be able to differentiate between both species.
3. Optimize your staining panel. If you do attempt a multi-species staining protocol, you may have 8 or more different colors in use in your staining panel. It will be critical to assure that your panel can be detected on a given flow cytometer and you will need to avoid using fluorescent stains that may have spectral overlaps and be difficult to detect accurately from one another. Pilot experiments are critical to working out these critical details.

Multi-species flow cytometry experiments may seem daunting, but can be a practical and efficient way to run samples and collect data. Consider working with flow cytometry experts such as contract research organizations to develop a sophisticated panel for such needs.