Flow cytometry is a powerful technique for multiparametric single-cell analysis. Here, we look at the basic principles of flow cytometry and key steps in the sample preparation workflow, and suggest ways to improve the quality of your data.
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Flow cytometry principles
During a typical flow cytometry experiment, a single-cell suspension is stained with fluorophore-labeled antibodies for specific cellular markers. The cells are then introduced into the flow cytometer, where a fluidics system directs them in single file past an interrogation point. Here, one or more lasers are focused, each producing a single wavelength of light at a specific frequency. When a cell crosses the interrogation point, the laser light is scattered and any bound fluorophores emit a fluorescent signal. The resultant light is directed by a series of dichroic mirrors and filters toward the instrument’s detectors. These are usually photomultiplier tubes (PMTs), which amplify the light and convert it into a measurable electrical signal. By analyzing the data with the use of appropriate experimental controls, researchers can identify distinct cellular populations.
Sample preparation for flow cytometry
Sample preparation for flow cytometry involves several main steps, which can vary depending on the experiment. If the aim is to detect only cell surface markers, the workflow involves processing the sample into a single-cell suspension, followed by blocking, immunostaining, and washing. However, if a combination of extracellular and intracellular markers is required for cellular identification, it is common to perform sequential staining. In this scenario, the samples are stained for cell surface markers before being fixed, permeabilized, and stained for intracellular targets, as shown in Figure 1.
Figure 1. Sequential staining for flow cytometry. Image provided by Fortis Life Sciences.
According to Amber Miller, Ph.D., Flow Cytometry Scientist at Fortis Life Sciences, sequential staining prevents cell surface epitopes from being compromised by certain fixatives. “Paraformaldehyde fixation can cause epitope masking, while methanol fixation can lead to changes in protein conformation,” she says. “Sequential staining helps to ensure that cell surface markers are not adversely affected by these types of reagents.”
Tips to improve the quality of flow cytometry data
There are many steps researchers can take to safeguard the quality of their flow cytometry data. Some key points to consider include the following:
- Optimizing sample preparation
Each of the steps involved in sample preparation requires careful optimization. Veronica Bruce, Ph.D., Scientist at Sartorius, suggests that before running important samples, researchers think through the experiment to ensure the resultant data is sufficient to test the hypothesis. “Critically, you will want to determine whether you are using the right antibodies, fluorophores, auxiliary reagents, and protocol to accurately characterize your sample,” she says. “This should include considering measures to reduce background and minimize non-specific binding, such as using excess protein in buffers, blocking Fc receptors, and excluding non-viable cells from data analysis.”
- Instrument setting controls
Every flow cytometer has its own set of quality control (QC) checks that should be performed on a regular basis. These serve to ensure that data is collected in a standardized manner while reducing operator-introduced variation. “Such checks typically cover laser alignment, fluidic stability, and detector performance,” explains Garret Guenther, Ph.D., Senior Product Manager for Flow Cytometry within the Cell Analysis Division at Agilent Technologies. “Automatic tracking of the QC test results over time using Levey-Jennings plots allows the user to compare results from day to day, or even over a long period, to determine if any measurements are trending in the wrong direction. This can prevent a possible issue before it impacts an important experiment.”
Bruce adds that prior to an experiment, instrument settings must be optimized for the data being acquired. “Conventional flow cytometers require detector voltage adjustments to improve signal resolution,” she says. “However, instruments like the iQue® Advanced Flow Cytometry Platform have a wide dynamic range that permits operation of the system without such adjustments being necessary for optimal performance. To make this process more robust, we implement daily quality control for each detection channel using fluorescent validation beads that assess mean fluorescence and CV against a set of standards. This practice verifies that instrument performance is within specification and can also be a useful tool in diagnosing issues if they arise.”
Careful use of controls can ensure that even the most complex experiments yield high integrity data. While the types of controls that are required for flow cytometry will be dictated by the experiment being performed, they generally include the following:
Biological controls—these are specific to the cell model and are used to verify that any observed changes are due to biological processes; they include diseased versus healthy cells, and stimulated versus untreated samples.
Unstained controls—useful for identifying autofluorescence, which can vary based on cell type, these are produced by omitting primary and secondary antibodies when immunostaining.
Single-color controls—these allow researchers to detect spectral overlap and apply compensation when performing multicolor flow cytometry experiments; they consist of samples that are stained with just one of the fluorophores in the panel.
Fluorescence minus one (FMO) controls—multicolor flow cytometry is susceptible to spillover spread (alterations in fluorescence due to the presence of other fluorophores); FMO controls are samples that are stained with all but one of the fluorophores used in the multicolor panel and help to determine the boundaries for background signal in the unlabeled channel.
Isotype controls—these serve to identify non-specific antibody binding; they consist of antibodies that share the same isotype as target-specific antibodies, and are labeled with the same fluorophore, but that do not recognize any target.
The use of automation for flow cytometry can be understood in various ways, but the over-riding goal is to improve workflow efficiencies and experimental reproducibility. “There is the automation of instrument functions, such as automated start up and shutdown, automated cleaning and rinsing procedures, and automated QC,” reports Guenther. “There is also automated sample collection, with the use of an autosampler, and then there is robotic automation to load plates onto an autosampler and allow for continuous running without human intervention. On the data analysis side, automation is being applied for tasks including statistical analysis and reporting.”
The level of automation that can be introduced into flow cytometry workflows will differ between labs. “One of the easiest ways to incorporate automation is to switch from using traditional flow cytometry tubes to using plates,” comments Miller. “With plates, you can more readily use multichannel pipettes to increase the speed of adding reagents and washes. Also, provided your flow cytometer can handle plates, this approach lets you automate sampling by setting the voltages for individual wells, which may each contain a different sample. These concepts can be similarly performed using a tube carousel, which can automatically move from tube to tube.”
Bruce notes that one of the best cases for automation is in the drug discovery process. “Given the abundance of potential chemical and biological treatments, automating sample preparation for therapeutics screening is a useful tool in harnessing the specificity and accuracy of flow cytometry while reducing bottlenecks in speed and throughput,” she says. “Overall, implementation of automated systems increases productivity and speeds up discovery in a precise and controlled manner that yields consistent results while minimizing the introduction of human error.”
Available tools and resources
A broad range of tools and resources is available to simplify the design and execution of flow cytometry experiments. These include Biocompare’s Flow Cytometry Panel Builderand a growing number of Optimized Multicolor Immunofluorescence Panels (OMIPs), as well as various open-access eBooks, webinars, and training courses. And of course companies such as Agilent Technologies, Fortis Life Sciences, and Sartorius are always willing to offer practical guidance that can streamline your research.