A Guide to Flow Cytometry

April 2, 2024

Flow cytometry is a fluorescence-based detection technique used in cellular research and diagnostics to analyze large cell populations at the single-cell level. Its high-throughput method can be used to investigate multiple cellular parameters at a time, including cell size, complexity, and the expression of specific target markers. By delivering rapid and quantitative insights into cellular properties, flow cytometry continues to be an indispensable tool in research, clinical, and industrial fields. Common applications for flow cytometry include immunophenotyping, cytokine detection, cell proliferation assays, apoptosis assays, and cell sorting. In this guide, we outline the key components of flow cytometry, including instruments, reagents, antibodies, and valuable resources to help ensure successful experiments and high-quality results.

Flow Cytometry: A Buyer’s Guide

Our in-depth eBook provides a broad overview of widely used flow cytometers currently on the market, highlighting key specifications and features to support informed decision-making, along with advice from some leading manufacturers. It includes a detailed specification sheet covering over 50 cytometers.

Flow cytometry basics

The workflow of a typical flow cytometry experiment proceeds as follows. The sample of cells in suspension is stained with fluorophore-conjugated antibodies targeting specific cellular markers. This sample is then introduced into the flow cytometer, where a fluidics system arranges the cells in a single-file stream as they pass through a laser interrogation point. As each passing cell moves through the laser beam, the light is scattered, and any attached fluorophores emit fluorescence. A system of dichroic mirrors and optical filters directs this emitted light toward the instrument’s detectors, which convert light into data-readable electrical signals. By analyzing the collected data alongside appropriate experimental controls, researchers can distinguish and characterize different cell populations.

Instrumentation

The flow cytometer is a specialized analytical instrument that has three key components: fluidics, optics, and data analysis. The fluidics system controls the movement of the sample through the instrument and determines attributes such as sample formats, fluid consumption, and flow rate. The optics component consists of excitation lasers, flow cell cuvettes, mirrors, fiber optics, filters, and detectors, all of which enable signal detection. Finally, data analysis involves processing light signals and converting them into electronic data for interpretation by the software.

Beyond the consistency of these core components, flow cytometers vary significantly in design, functionality, and price. Key differences include laser configurations, optical filters, detection systems, sample loading formats, and analysis software, which can differ across models and manufacturers. Entry-level models with fewer lasers typically range from $100,000 to $250,000, while high-end systems with six or more lasers can exceed $500,000. Flow cytometers are a significant investment and require careful consideration when comparing different models. The maximum number of parameters or fluorescent channels is an important specification, as this will dictate the complexity of experiments that the instrument can accommodate. This is also directly influenced by the number of lasers the system can equip. Other useful considerations include laser wavelengths, sample formats, additional functions, and compatibility with automated workflows.

Flow Cytometers Search Tool

The Biocompare flow cytometer search tool is a comprehensive catalog that lists instruments across different manufacturers. Use filters to easily compare equipment specifications to find the cytometer that best fits your research needs.

Sample preparation

The process of preparing samples for flow cytometry consists of several key steps that may differ based on the experiment's objectives or the nature of the sample. A protocol will generally begin by collecting samples and processing them into a suspension. A blocking solution is added to prevent the non-specific binding of antibodies to exposed proteins and antigens. The sample is washed and then incubated with primary antibodies specific to the proteins, antigens, or subcellular components of interest.

If both extracellular and intracellular markers are targets in the experiment, the protocol will also include steps for fixation and permeabilization. In this case, steps are often done sequentially so that cell surface protein epitopes will not be compromised by the masking effect of fixatives.

Depending on the sample, specialized buffers or additional treatment may be necessary to optimize the experiment. For blood samples, red blood cell lysis buffers can eliminate unwanted RBCs when analyzing leukocytes. Blood fixatives or stabilizers may also be used for fresh blood samples that need to be preserved, such as during prolonged transport. Specialized kits containing complete reagent sets can help streamline flow cytometry experiments, such as assays for cell viability, proliferation, or apoptosis. Read our articles that highlight the general flow cytometry protocol and offer more tips for sample prep.

Antibody staining

Antibody staining is a crucial procedure within flow cytometry sample prep, as this drives the detection of specific cellular targets. Staining can be performed directly using fluorophore-labeled primary antibodies or indirectly using unlabeled primary antibodies paired with a labeled secondary antibody. A panel consisting of multiple antibodies can be used on a single sample for a multi-parameter experiment detecting several cellular markers.

Careful antibody selection will be necessary for effective staining and meaningful results. First, the primary antibody must be appropriately selective to the target epitope to ensure robust and accurate signals. Antibodies can perform differently across immunodetection applications, so it is important to choose those validated for flow cytometry. Other antibody considerations include clonality (monoclonal, polyclonal, or recombinant), fluorophore labels, and host species. Isotype control antibodies should also be considered for certain cell types. Read our guide to flow cytometry antibody selection for more useful information.

Flow Cytometry Panel Builder

Careful planning of a multicolor flow cytometry experiment is crucial to ensure robust resolution, sensitivity, and biological accuracy. Our tool lets you design your flow panel using instrument configurations based on any flow cytometer of your choice.

High-parameter flow cytometry

Most users of conventional flow cytometry typically measure less than 12 markers simultaneously. Measuring upwards of 20 markers begins to fall in the realm of high-parameter cytometry, which is useful for in-depth characterization of immune cell subsets, signaling pathways, and rare cell populations. Increasing the number of parameters becomes increasingly difficult due to several limiting factors, such as instrument capabilities and spectral overlap. Generally, the greater the number of detectors, the better equipped a cytometer will be in performing high-parameter analysis.

Spectral flow cytometry employs specialized detectors to capture the full fluorescence spectrum of fluorophores across the entire visible wavelength range. By measuring the unique spectral fingerprints of each fluorophore, it can sufficiently distinguish very closely emitting fluorophores, overcoming the limitations of spectral overlap. This technology supports vastly more of fluorophores in a single experiment, enhancing panel design flexibility and expanding the scope of multiparametric analysis.

Cell sorting

A fluorescence-based cell sorter is, in essence, a flow cytometer that can both analyze and separate cells for collection. All flow cytometers share the same core components—fluidics, optics, and electronics—whether they are designed solely for analysis or equipped for cell sorting. Standard flow cytometers specialize in analyzing cells, often with many parameters, but discarding them after. In contrast, cell sorters, while analyzing cells in a similar manner, specialize in separation and collection, often handling the sample under more gentle conditions to ensure viability. This capability unlocks a wide range of applications, including single-cell genomics, proteomics, cell-based assays, and immunotherapy.