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Flow cytometry enables researchers to label proteins of interest using fluorophore-conjugated antibodies and other fluorochromes. Much like immunohistochemistry, which produces two- or three-dimensional images of tissue, flow cytometry allows researchers to assess cell-associated protein expression. This can be used to identify the frequency and numbers of a cell type within a sample or levels of expression of a protein of interest. Flow cytometry experiments have been used extensively in the preclinical to clinical pipeline, by allowing researchers to assess the immune response to therapeutics by evaluating the cellular phenotype before and during a course of treatment.
Flow cytometry workflows
Flow cytometry can be used to analyze a variety of different sample types for both in vivo and ex vivo studies. In a flow cytometry workflow, tissue, blood, or other sources of cells are isolated and processed into single cell suspensions containing the cells of interest. The cells are then labeled with cell viability dyes or fluorescent antibodies. Samples can then be analyzed live or fixed, allowing for later analysis, on a flow cytometer. Using commercially available software including FlowJo or FCS Express, cells can be measured for size (forward scatter), granularity (side scatter), cell viability, and cell surface or intracellular protein expression.
Preclinical research relies on humanized mice
Effective mouse models that recapitulate human disease are key in advancing preclinical research. Super-immunodeficient mice as well as advanced humanized models have enabled a deeper understanding of disease mechanisms and accelerated the development of novel therapeutics that have a greater chance of success in clinical trials. Humanized models are generated from the myeloablation and subsequent HSC or PBMC engraftment of a super-immunodeficient mouse. Variability between mouse models, along with other factors, contributes to a differing amount of engrafted immune cells in the resulting model. In order to fully understand the immune cell composition of a humanized mouse, it’s necessary to test the blood and tissues of interest and characterize the cell types present. Utilizing flow cytometry on these samples enables:
- Quality control of successful tumor engraftment in humanized models
- Randomization of animals before study conception
- Monitoring of immune cell reconstitution
- Characterizing cell populations, including cell proliferation and activation
- Endpoint analysis
- Tumor-infiltrating lymphocyte analysis
Many technical advancements have improved how flow cytometry is conducted, including improved flow cytometers for higher parameter analysis, refined intra- and extracellular staining protocols, and best-in-class antibodies. Still, human error, small differences in sample integrity, and characterizing rare cell populations all require careful consideration during project design and data analysis.
Despite how widely flow cytometry is used with humanized mice, there are no standardized protocols, and this can create confusion.
Taconic’s experts can help discuss a wide range of topics related to flow cytometry and humanized immune system mice including: design of flow cytometry panels (including assessment of the myeloid compartment), important considerations for blood sampling during humanized mouse experiments, chimerism calculation methods, application of spectral flow cytometry to humanized mouse analysis, and the types of data provided with shipment of Taconic humanized immune system mice.
Interested in learning more about flow cytometry for humanized mice? Connect with one of our experts today taconic.com/gen.
