Flow cytometry has various uses that can greatly improve scientific research and the development of drugs and therapeutics. These uses can be for providing additional details such as the mechanism of action or cytokine profiling. They may also be fundamental for the research such as sorting out the cells needed for the experiment or determining the immunological effects of treatment. The many applications of flow cytometry have resulted in academia, biotech, pharmaceutical, and clinical labs all taking advantage of this technology.
AML Gating Strategy: Leukocytes are selected by forward- versus side-scatter. Singlets are identified based on the linear relationship of forward-scatter area and height parameters. Viable cells are gated based on the dim staining with Live/Dead viability dye. From the live cell gate, total CD45+ events are selected, as well as a permissive CD45+ AML gate, from which AML cells are selected for expression of CD34 or CD117, or both (red arrow). From CD45+ SSClow, CD3+, CD19+ and CD56+ cells are identified (blue arrows).
The most common usage of flow cytometry is immunophenotyping. Immunophenotyping allows for mixed cell populations to be identified for the presence and proportion of specific cells of interest. Single or multiple cell markers may be analyzed to identify specific cells. Additionally, using a combination of surface and intracellular staining can allow for more specific information on the cell population. One example of immunophenotyping in the characterization of different T cell subsets such as identifying CD4+ helper T cells, CD8+ cytotoxic T cells, or antigen-specific T cells (1), which can provide valuable information for immunological studies and disease treatments involving autoimmunity, cancer, and others. Another valuable usage is for leukemia/lymphoma diagnosis. Through using a well-designed antibody panel, flow cytometry allows for blast identification and characterization, lineage assignment, rare abnormal cell detection, and detection of therapeutic targets (2).
2. Cell Health and Growth
Determining the phase of the cell cycle, viability, proliferation, and cell death mechanisms are all other applications of flow cytometry. One of the first applications of flow cytometry was cell cycle analysis since DNA binding dye and protein expression can differentiate cells containing higher DNA content and synthesis of proteins of cells preparing for division (3). The cell cycle can provide crucial information in numerous areas of research.
Additional important measurements in many toxicological studies and clinical trials are determining changes to cell viability and proliferation. Furthermore, an important cellular pathway that regulates homeostasis, proliferation, and cell growth called autophagy can be measured with flow cytometry (3). Flow cytometry can also determine the mechanisms of cell death. This includes differentiating between necrosis and early apoptosis with markers like Annexin V, and others including caspase activation, pyroptotic cell detection, and blast cell Bcl-2 expression (3,4). The ability to measure these in the whole population and in cell subsets gives an advantage to using flow cytometry over other techniques.
3. Sorting of Cells and Particles
Cell sorting is another highly valuable function of flow cytometry. This allows for the selection of specific cell types from mixed populations. Some of the most common uses of this technique are purifying transfected cells, stem cells, tumor cells, lymphocyte subpopulations, and tumor-infiltrating lymphocytes for further analysis (5). The selection and purification of these cells can be used for non-invasive patient monitoring, personalized medicine, clinical studies, and non-clinical research.
There are various other uses in addition to sorting human and animal cells. These sorting capabilities can be applied to filamentous fungi (6). Bacterial isolation for researching transcriptional heterogeneity and antibiotic resistance is another use (7). Flow cytometry sorting can provide a high-throughput method for the purification of axenic cultures of microalgae, which is being examined as a potential green alternative for food, feed, and fuel production (8).
In addition to selecting cells, flow cytometry has applications for sorting particles. Recent developments with nanoFACS and flow virometry methods allow for the sorting of biological nanoparticles including extracellular vesicles and small viruses (9). Unlike other nanoparticle purification techniques, nanoFACS and flow virometry can rapidly sort from mixtures without the need to first concentrate the sample and produces a product that retains its biological activity (10).
4. Serological Testing
Testing for the presence of antibodies or serological testing for the diagnosis of infectious diseases is an important part of many clinical and research studies. Using flow cytometry for serological testing is more sensitive than traditional immunoassays, allows for high throughput, and can measure multiple antibodies simultaneously (11). For example, an early and accurate diagnosis of the severe parasitic disease visceral leishmaniasis can provide lifesaving treatment. Flow cytometry has been identified as a more sensitive and specific method to diagnose visceral leishmaniasis in canines (12) and HIV-infected patients (13).
The use of flow cytometry for SARS-CoV-2 diagnosis is another example. Unlike many other commercial SARS-CoV-2 serological tests, flow cytometry can detect multiple antibodies, which can give a clearer evaluation of the infection and the quality of the immune response. The high sensitivity of flow cytometry can also help identify the severity of the infection and differentiate between vaccinated and infected individuals (14). Overall, the use of flow cytometry for serological testing provides a highly sensitive and high throughput method that has many uses.
5. Other Biological Applications
In addition to the previously mentioned applications, there are quite a few other biological uses for flow cytometry. Information about cellular characteristics including structural and chemical can be measured from mammalian cells, bacteria, yeast, and viruses. This includes analyzing organelles, extracellular vesicles, nuclei/DNA, chromosomes, and cell signaling events (3). Some examples of cell signaling that can be quantified include reactive oxygen species (ROS) detection and intracellular calcium flux (3). These are significant measurements for clinical outcomes such as DNA damage (15) and the progression of diseases like lymphocytic leukemia (16).
There are also other applications. Antigen-specific responses can be determined with quantification of extracellular and intracellular cytokine/chemokine production or with testing for cell recognition, which is a necessary analysis for vaccine development (5). Flow cytometry has been widely used for studying phagocytosis with applications in clinical studies of human immunodeficiencies, veterinary, and environmental settings (3). Flow cytometry can also allow for simultaneous measurement of proteins and mRNA expression through a combination with fluorescent in situ hybridization (FISH) (3,5).
6. Drug Development
Flow cytometry techniques have been a valuable tool for drug development with the advantage of enabling multiplexed phenotypic primary screening of cells and screening of sources for biological drugs like antibodies (17). For example, flow cytometry is of critical importance in nearly every stage of CAR T therapy development. This includes patient diagnosis, characterization of the manufactured product (e.g. immunophenotyping to determine cellular composition and purity), and post-infusion (e.g. functional state and target antigen loss) (18).
Mechanism of action can also be assessed using functional assays, cytokine profiling, and immunophenotyping. For clinical studies using biotherapeutics, receptor occupancy (RO) assays can add value to each stage of drug development. RO assays are measurements of the binding of the therapeutic to its cellular target. RO assays can help preclinically model starting dose, demonstrate target binding in Phase 1 clinical trials, determine efficacious dosing range in Phase 2, and define population pharmacokinetic characteristics in Phase 3 (19). Overall, there are many applications for flow cytometry throughout the whole process of drug development.
In conclusion, there are many diverse applications for flow cytometry. Numerous endpoints can be analyzed such as with immunophenotyping or cell apoptosis determination. Cells can also be sorted for further downstream analysis or treatments. There are many advantages to this technique including high specificity, high throughput, and multiparameter abilities. Flow cytometry has been applied in various areas including molecular biology, immunology, virology, infectious diseases, and cancer biology.
- Mousset, Charlotte M., et al. "Comprehensive phenotyping of T cells using flow cytometry." Cytometry Part A 95.6 (2019): 647-654.
- DiGiuseppe JA, and Wood BL. Applications of Flow Cytometric Immunophenotyping in the Diagnosis and Posttreatment Monitoring of B and T Lymphoblastic Leukemia/Lymphoma. Cytometry Part B 2019; 96B: 256–265.
- Cossarizza, Andrea, et al. "Guidelines for the use of flow cytometry and cell sorting in immunological studies." European journal of immunology 49.10 (2019): 1457-1973.
- Mestrum, Stefan GC, et al. "The potential of proliferative and apoptotic parameters in clinical flow cytometry of myeloid malignancies." Blood Advances 5.7 (2021): 2040-2052.
- McKinnon KM. Flow Cytometry: An Overview. Curr Protoc Immunol. 2018;120:5.1.1-5.1.11. Published 2018 Feb 21. doi:10.1002/cpim.40
- Bleichrodt, Robert-Jan, and Nick D. Read. "Flow cytometry and FACS applied to filamentous fungi." Fungal Biology Reviews 33.1 (2019): 1-15.
- Imdahl, F., Vafadarnejad, E., Homberger, C. et al. Single-cell RNA-sequencing reports growth-condition-specific global transcriptomes of individual bacteria. Nat Microbiol 5, 1202–1206 (2020). https://doi.org/10.1038/s41564-020-0774-1
- Pereira, Hugo, et al. "Fluorescence activated cell-sorting principles and applications in microalgal biotechnology." Algal research 30 (2018): 113-120.
- Morales-Kastresana, Aizea, et al. "High-fidelity detection and sorting of nanoscale vesicles in viral disease and cancer." Journal of extracellular vesicles 8.1 (2019): 1597603.
- McNamara, R.P., Dittmer, D.P. Modern Techniques for the Isolation of Extracellular Vesicles and Viruses. J Neuroimmune Pharmacol 15, 459–472 (2020). https://doi.org/10.1007/s11481-019-09874-x
- Jani, Ilesh V., et al. "Multiplexed immunoassays by flow cytometry for diagnosis and surveillance of infectious diseases in resource-poor settings." The Lancet infectious diseases 2.4 (2002): 243-250.
- Ker, H.G., Coura-Vital, W., Valadares, D.G. et al. Multiplex flow cytometry serology to diagnosis of canine visceral leishmaniasis. Appl Microbiol Biotechnol 103, 8179–8190 (2019). https://doi.org/10.1007/s00253-019-10068-x
- Da Silva, Elis D., et al. "A Flow Cytometry-Based Serological Assay to Detect Visceral Leishmaniasis in HIV-Infected Patients." Frontiers in Medicine 8 (2021).
- Cáceres‐Martell, Yaiza, et al. "Single‐reaction multi‐antigen serological test for comprehensive evaluation of SARS‐CoV‐2 patients by flow cytometry." European journal of immunology 51.11 (2021): 2633-2640.
- Durdik, Matus, et al. "Microwaves from mobile phone induce reactive oxygen species but not DNA damage, preleukemic fusion genes and apoptosis in hematopoietic stem/progenitor cells." Scientific Reports 9.1 (2019): 1-12.
- Heitmann, Jonas S., et al. "A novel flow cytometry‐based assay to measure compromised B cell receptor signaling as a prognostic factor in chronic lymphocytic leukemia." Journal of leukocyte biology 108.6 (2020): 1851-1857.
- Edwards, Bruce S., and Larry A. Sklar. "Flow cytometry: impact on early drug discovery." Journal of biomolecular screening 20.6 (2015): 689-707.
- Sarikonda, Ghanashyam, et al. "Best practices for the development, analytical validation and clinical implementation of flow cytometric methods for chimeric antigen receptor T cell analyses." Cytometry Part B: Clinical Cytometry 100.1 (2021): 79-91.
- Stewart, Jennifer J., et al. "Role of receptor occupancy assays by flow cytometry in drug development." Cytometry Part B: Clinical Cytometry 90.2 (2016): 110-116.
Joella Wu is passionate about scientific research in areas of immunity, virology, microbiota, and diseases. She received her undergraduate degree in Biology and PhD in Toxicology from the University of Georgia. Her dissertation research focused on endocrine disruptors and their effects on the gut microbiota, immunity, and type 1 diabetes. After graduating, she did a postdoc at Emory University in a drug discovery lab focused on discovering new therapies for HIV+ patients. She also worked on developing an assay for quick discovery of new drugs effective against SARS-CoV-2. After her postdoc, she started working in a clinical lab at Tempus where she was involved in running various assays such as diagnostic assays for SARS-CoV-2 and metabolic genotyping assays. Currently, she is a Senior Scientific Data Analyst at FlowMetric. In her current position, she utilizes her previous experience in flow cytometry to provide insights for clients on their research and clinical studies.