The first steps towards the creation of flow cytometry can be traced back to the revolutionary Coulter principle detailed in Wallace H. Coulter’s 1953 U.S. Patent No. 2,656,508. This proposed a method for studying particle size and dynamic based on electrical impedance proportional to the volume of analyte traveled through a channel. Mack Fulwyler further expanded on this concept with his 1965 cell sorter prototype, which integrated cell separation techniques to accommodate specific sizes for collection. Since the creation of these key innovations, many improvements have been made to advance flow cytometry’s capabilities in basic science research and clinical applications.
Flow Cytometry Basics
While there are many flow cytometry techniques, the majority follow the same general protocol. First, cells are stained with reagents that closely associate with an intended cell process or structure while simultaneously emitting some reporter signal. A popular choice is using one or more antibodies conjugated to organic fluorescent molecules such as Alexa Fluor 488, Cy5, or Texas red. Cell samples can also be transfected to co-express fluorescent proteins such as GFP, which has become the most popular along with its family of mutation derivatives. Transient mechanisms such as oxidative stress or autophagy may benefit from
fluorescent probe technologies as a means to monitor its progress over time. For more information on fluorochrome excitation and emission spectra, please see the online
spectra viewer for flow cytometry.
Figure 1. Spectra Viewer depicting fluorescence spectra profiles for Alexa Fluor 488 and Enzo’s Necrosis detection reagent (ENZ-51002).
Alexa Fluor 488 - (Ex: 490 nm, Em: 525 nm), Enzo’s Necrosis detection reagent (ENZ-51002) – (Ex: 546 nm, Em: 647 nm)
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Stained cells are then fed into fluidic systems, which inject pressurized cell samples in buffered solutions through a sheath flow one by one. Samples reach an interrogation point and are subjected to a system of lasers and photon collection optics that generate visible and fluorescence signals from reporter molecules. This light passes through dichroic filters, where signals of specific wavelengths are steered to be read by appropriate detectors.
Each analyzed sample generates a data point, or event. Compiled events can then be gated, or manually separated into groups based on desired spectral properties and scanning patterns. For example, the forward scatter scans along the path of the laser and indicates the relative size of cells. Side scatter analyzes samples 90° relative to the laser for internal complexity and granularity of cells. While the parameters for gating have been limited in the past, today’s flow cytometers have evolved to analyze samples with up to 32+ parameters and flow rates of 100,000 cells/sec.
Different Methods of Flow Cytometry
FACS
Building off the work of Coulter and Fulwyler, Len Herzenberg developed fluorescence-activated cell sorter (FACS) in the 1970s. FACS differs from conventional flow cytometry due to its ability to sort and accurately purify specific cell populations at a high efficiency. Using fluorescence as its primary mode of selection, identified sample liquid streams are oscillated into drops and are given positive or negative charges. These charged droplets pass through metal deflection plates to be directed in collection vessels of interest (plates, slides, tubes, etc). This can be incredibly useful for characterizing and sorting heterogeneous sample matrices, such as blood containing a diverse number of lymphocytes and red blood cells of different maturity.
Figure 2. Jurkat cells were induced with 100 µM pyocyanin (general ROS inducer, panel B), 200 µM antimycin A (superoxide inducer, panel C) or 1 µM of t-butyl-hydroperoxide (peroxide inducer, panel D), stained with our two color ROS-ID® Detection Kit and analyzed using flow cytometry. Untreated cells (panel A) were used as a control. Cell debris were ungated and compensation was performed using single stained pyocyanin-treated samples. Red numbers reflect the percentage of the cells in each quadrant.
Featured product: -ID® Total ROS/Superoxide detection kit (cat # ENZ-51010)
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Despite FACS’ versatile application, cells often undergo intensive stresses that impact sample viability after processing. Sorting mechanisms subject samples to high pressures as cells push through narrow tubes. Researchers should consider larger nozzles that are at least 5x the estimated diameter of their cells to reduce this strain. Some cells may be more delicate than others and require lowered flow rates to minimize shear forces. Samples also face unwanted impact forces from being propelled at high velocities during droplet formation and collection. Adding collection mediums or buffers in collection devices should always be considered to reduce this stress.
Imaging Flow Cytometry & Mass Cytometry
In addition to FACS, flow cytometry has been adapted to incorporate other technologies to generate more specific and informationally useful techniques. Imaging flow cytometry (IFC) combines the high throughput nature of conventional flow cytometry with the imaging power of fluorescence microscopy. IFC differs from conventional flow cytometry as it can analyze many additional unique parameters such as cell shape, texture, and morphology. Combining this data creates a composite profile of each cell and allows for greater specificity in population grouping. This level of detail is especially useful when examining cell-cell interactions, rare cell types, and transition states. To see an application of IFC, please read our
application note that discusses how the
PROTEOSTAT® Protein aggregation assay contributes to the development of protein-based drugs.
Mass cytometry is another interesting composite application that combines “time-of-flight” mass spectroscopy. Although this process shares workflows similar to conventional flow cytometry, it does not use fluorescent reporter molecules. Mass cytometry opts to label with heavy metal ion-tagged antibodies or rhodium- and iridium-conjugated DNA intercalators. Metal signals are then analyzed through time-of-flight mass spectrometry and integrated on data matrices with each row representing a single mass scan by detectors. With no light detection, mass cytometry benefits from having no
fluorescence emission spectral overlap and minimizes background. However, no functional studies can be conducted on processed cells as they are effectively destroyed from being atomized and ionized.
Flow Cytometry in Basic Science Research and Clinical Applications
Applications in Immunology & Hemotology
Traditional methods for quantifying and identifying blood cells use blood smears, which spreads blood on slides thin enough such that cells can be visually counted and differentiated after staining. Although this method is still used to this day, it is both tedious and lacks precision. Immunophenotyping is a superior method by which heterogeneous cell samples undergo multi-parameter analysis to identify specific protein expression profiles. Detected antigens generally fall into two categories, cell surface markers or intracellular components.
Cell surface marker staining is generally the easier of the two and also utilizes fluorochrome-conjugated antibodies that target specific structures. Most lymphocytes can be identified from a combination of different “cluster of differentiation” (CD) antigens expressed on surfaces. T-cells are known to have high
CD3,
CD4, and CD8 expression while B-cells have high CD19 and CD20. Surface marker staining can also be used to distinguish disease states as certain cell pathologies express markers at different magnitude depending on its disease progress. A notable example is J.V Georgi’s 1987 study on the relationship between HIV infection and CD4 expression in T-cells. These findings demonstrated decreased CD4+ expression and lymphocytic proliferation being associated with longer incubation with HIV and advanced AIDS progression.
Table 1. Cluster of differentiation (CD) marker profiles for various lymphocytes
Immunophenotyping for intracellular markers is more complex as cells first need to be treated with appropriate agonists to upregulate target analytes to detectable levels. Samples are then fixed and permeabilized to allow for fluorochrome-tagged antibody infiltration and analyte staining. Several popular intracellular targets are proliferation markers such as
Ki-67 or intracellular regulation of cytokines such as
IL-33. These two divisions of immunophenotyping prove incredibly useful for today’s physicians to quickly identify sample results and anticipate future treatment plans.
Although combining intracellular staining and surface staining does hold high practical value, researchers need to be wary of several challenges. Due to intracellular staining requiring harsh buffers for fixation and permeabilization, this technique may result in reduced signal intensity and inadequate antibody binding to surface markers. While there is no universal buffer that can accommodate both types of staining at this time, new fixation and permeabilization buffers are constantly being developed to overcome this challenge. Utilizing these two areas of immunophenotyping provides physician’s valuable information on blood samples and aids in future action plans for treatment.
Genomics
Flow cytometry applications in genomics studies are numerous, with an especially high focus placed on monitoring cell cycle phases. Researchers have traditionally avoided using fluorochrome-conjugated antibodies. Instead, cell cycle phases are identified through changes observed from staining patterns from fluorescence DNA intercalating agents such as
propidium iodide and
acridine orange. With increased fluorescence correlating with increased DNA content, researchers may also use this metric as a way to screen for aneuploidy or abnormal DNA content in sample populations. Within a clinical context, this is useful for screening leukemia as well as other malignancies.
Figure 3. Effect of a cell cycle perturbation agent on DNA synthesis in Jurkat cells. The cells were treated with 0.1µg/ml Nocodazole for 20 hours. Cells were then washed and stained with NUCLEAR-ID® Red Cell Cycle Detection Reagent.
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Researchers and clinicians may also opt to co-stain RNA as a means to further validate cell states and pathologies. RNA dye stains follow a similar principle of helical intercalation, with popular dyes including pyronin Y and
7AAD. Differences in RNA staining intensity is exhibited between cell cycle phases, with lower RNA levels associated with dormant G0 and higher staining with G1, S, and G2/M proliferation. In some cases, researchers may find it more desirable to implement
ISH probe technologies against RNA as a metric for protein expression. This is particularly useful when researchers have difficulty finding antibodies for the target protein and/or the price exceeds that of one’s budget.
Cell Viability and Death
Within the context of cell viability, flow cytometry is also valued for its applications in proliferation assays. Cells are generationally tracked by being stained with inheritable permanent dyes that cross cell membranes and bind to intracellular structures of interest. Daughter cells inheriting these dyes display decreased fluorescence signal per replicative event. To accommodate this type of study, we offer the
CYTO-ID® Red long-term cell tracer kit and
CYTO-ID® Green long-term cell tracer kit allows for staining lasting up to 96 hours with minimal transfer of fluorescence from dye-labeled to unlabeled cells. Additionally, we offer the NUCLEAR-ID® Blue/Red cell viability reagent (GFP-CERTIFIED®) that produces high-quality staining pattern separation between live and dead cell populations.
Figure 4. Flow cytometry analysis of fluorescence of mixed population of Jurkat cells over time. Jurkat cells stained with CYTO-ID® Green Tracer dye were mixed with an unstained population of Jurkat cells and incubated over a 120-hour period.
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Flow cytometry may also be applied to studies on intrinsic apoptosis, which is often attributed to DNA damage or ER stress. These downstream events lead to the activation of pro-apoptotic Bcl-2 family proteins as well as caspases. Researchers can take advantage of these events by monitoring the position of these molecular components as well as gauging enzymatic activity through the use of cell-permeable fluorogenic substrates. Apoptosis is also characterized by the rapid exchange of phosphatidylserine between inner and outer cell membranes. This offers another way to gauge apoptotic progress through reliable staining with reagents such as Annexin V. Please see the
GFP-CERTIFIED® Apoptosis/Necrosis detection kit, which readily distinguishes between healthy, early apoptotic, late apoptotic, and necrotic cells.
Mobile Flow Cytometry, an Interesting Proposition?
Although we have seen many reasons that support flow cytometry’s value, there are still limitations in its practicality. The most obvious relate to how large, expensive, and power-intensive these instruments can be. This limits their use in laboratories and leaves resource-poor areas devoid of their clinical benefit. These problems set the stage for the development of cheaper and more accessible platforms to conduct mobile flow cytometry.
At this time, no current mobile flow cytometers are available in the market for this intended purpose. The closest advancement in mobile flow cytometry is optic fluidic fluorescent imaging cytometry on cell phones with a spatial resolution of ~2 μm. However, relatively low flow rates of ~1 μL/min, limited sample rate (30 fps for most phones), and the inability to change lenses easily make these systems applications limited. Nevertheless, unique ideas are constantly being supported by multiple fronts to address this demand. In time, flow cytometry will have a larger role in mobile health and can expand health care opportunities to greater populations.
The Enzo portfolio offers a complete range of products for
flow cytometry, contains over 3,000 antibodies to detection kits to monitor
oxidative stress,
autophagy,
cell senescence, and much more! For more information on Enzo’s collaborative works in flow cytometry, please check our full list of
application notes on this subject. For any further questions and concerns regarding any of our products, please reach out to our
Technical Support team. We are here to assist you with your flow cytometry solutions!
