Standard flow cytometry protocols may require that you stimulate a batch of control cells with some sort of non-specific mitogen or antibody. Have you ever considered why these control samples are stimulated and what this tells you about your experiment?
1. You can check how well your cells are functioning. We all know that it’s important to stain cells with viability markers to distinguish between live and dead cells, but non-specific stimulation lets you confirm that your cells can indeed produce cytokines and express cell surface markers.
2. You can set up accurate flow cytometry gates. When you begin your flow cytometry acquisition you need unstimulated negative control cells, as well as your stimulated positive control cells, to help you set up appropriate parameters, or “gates,” in your flow cytometry software. This will insure that useful data will be collected for your experimental samples.
3. You can create a “mock” phenotype for assay development. Flow cytometry assay development requires having samples that you can evaluate to insure your stimulation and staining protocols are working correctly. Stimulated cells will have a “mock” phenotype that can be used to develop and validate assays under development.
Stimulated cells are a critical part to routine flow cytometry experiments as well as for use during assay development. Never forget this key component in your flow cytometry experiments!