Providing almost unrivalled statistical power in comparison with other commonly used techniques, flow cytometry delivers key insight into cell populations, even those of extremely low abundance. We recently asked three flow cytometry experts—Christopher Manning, group leader, flow cytometry, at Cell Signaling Technology; Alexandra Wittmann, senior scientist, flow cytometry, imaging team at Abcam; and Johannes Fleischer, global marketing campaign manager at Miltenyi Biotec—to share their recommendations for developing and implementing effective flow cytometry staining strategies. Here is what they had to say.
Prepare samples appropriately to enable specific binding to target antigens
“To achieve successful staining, it is important to prepare samples in a manner compatible with the antibodies used in the assay,” explains Manning. “Whether this means using live cells or fixed cells, cells permeabilized with detergents or solvents, the protocol is a critical factor governing antibody utility. Changes to reagents or conditions necessitate revalidation of the antibodies used in the assay, because while many antibodies are flexible and work well in a diverse array of protocols, some may only bind correctly under a specific set of conditions.”
Image: Jurkat cells were treated, fixed with 4% formaldehyde and permeabilized as indicated. One antibody maintains performance in multiple protocol conditions while one requires a specific protocol for optimal staining, highlighting the importance of following manufacturer-provided protocols where possible and the need for re-validating with any protocol variations. Image courtesy of Cell Signaling Technology.
“If your experimental protocols are too harsh, you could denature your target antigen so that your antibody no longer recognizes it,” notes Wittmann. “For example, if you are isolating cells from tissue, it can be wise to use a gentler cell dissociation reagent as an alternative to traditional enzymatic digestion with trypsin. When cells are already in suspension, it can be advantageous to use polypropylene tubes to avoid loss of cells that may adhere to the plastic.”
Fleischer adds that to remove the need for enzymatic digestion of tissue samples, researchers may instead choose to perform mechanical dissociation. “Our gentleMACS™ Dissociator uses pre-set programs and optimized tissue-specific dissociation buffers to preserve surface epitopes,” he says. “We’ve generated extensive testing data demonstrating that this approach improves experimental reproducibility.”
Include relevant controls
“Few other assays demand as much unsubstantiated faith in antibody performance as flow cytometry,” notes Manning. “Where western blots allow visualization of the molecular weight of the labeled protein, and immunocytochemistry and immunohistochemistry enable observation of protein localization, flow cytometry only provides cell-by-cell fluorescence. The use of proper controls to indicate whether an antibody is binding specifically is therefore critical for flow cytometry.”
“Depending on your experiment, relevant controls can include unstained, positive, negative, isotype, viability, Fc-blocking, fluorescence minus one (FMO), and compensation controls,” reports Wittmann. “For staining, it would be advisable to use positive and negative controls, such as wild-type and knockout samples. These are often complemented by isotype controls, viability stains, and Fc-blocking controls to inform background signal due to non-specific antibody binding.”
Key takeaways
- Prepare samples appropriately
- Include relevant controls
- Understand your flow cytometer
- Optimize the order of antibody addition
- Consider cell enrichment
- Select antibodies proven to work in flow
- Study existing protocols and literature
- Consider using directly-conjugated antibodies
- Protect fluorophores from exposure to light
- Incorporate automation to improve reproducibility
“To reduce the complexity of experimental planning, all our REAfinity™ Recombinant Antibodies are of the IgG1 isotype and can be used with our universal isotype control antibody (clone REA293),” reports Fleischer. “Furthermore, REAfinity Antibodies benefit from an engineered Fc region, which prevents antibody binding to Fc receptors and removes the need to include Fc-blocking controls.”
Commonly used viability stains such as DAPI and DRAQ5 rely on membrane integrity for distinguishing live cells from dead, meaning they can only be used for cells being surface stained. “More recently, fixable viability dyes such as the Ghost Dye™ reagents have become popular,” says Manning. “These permanently label dead cells prior to fixation, enabling live/dead discrimination even after cells have been fixed and permeabilized for analysis of intracellular proteins.”
“The FMO control is fundamental in multicolor experiments because it allows for correct gating to accurately identify positive cells,” explains Wittmann. “Compensation controls are also essential to experimental set-up, allowing spectral overlap to be approximated and subtracted from the total detected signal to yield an estimate of the actual amount of each dye.”
Manning adds that since many flow cytometry experiments are multicolor, it is sensible to choose fluorophores with minimal spectral overlap to reduce the need for fluorescence compensation. “Once you get above a handful of colors, it’s impossible to have truly non-overlapping profiles,” he says. “However, with an increasingly diverse range of fluorophores commercially available, it is possible to minimize compensation by distributing these across different lasers.”
Understand your flow cytometer
“It’s very important to familiarize yourself with your flow cytometer, its lasers, filters, and software,” says Fleischer. “This will help you to select appropriate fluorophores for your experiment and increase your chances of staining success. Although flow cytometry has long been a domain for wizards with the capability and experience to design great panels and run highly complex machines, it’s clear that intuitive and simplified design has been key to broader adoption of the technology. For example, our state-of-the-art online panel builder tool allows researchers to easily limit fluorochrome-conjugate options to match low- or high-abundance antigens, while our MACSQuant® Analyzer flow cytometers feature MACSQuantify™ Software for automated calibration and compensation. I strongly believe that automation and user-centric assistance in experimental design and analysis are making flow cytometry more accessible, leading to improvements in standardization and reproducibility.”
Additional strategies for success
According to Manning, another approach to generate meaningful flow cytometry data is to optimize the order of antibody addition for combined extracellular/intracellular panels. “It is possible to combine antibodies designed for live-cell phenotyping and fixed-cell intracellular readouts such as protein abundance, phosphorylation, or other signaling events,” he says. “The key is to determine whether the antibodies binding to CD markers or other cell surface antigens are compatible with the protocol used for intracellular staining. It is well worth the upfront time to test whether you can add all the antibodies together or whether you require separate live-cell and fixed-cell incubation steps, especially if you plan to use the same panel of antibodies across multiple experiments.”
“Cell enrichment can be beneficial, especially when working with rare cell types,” notes Fleischer. “This can be achieved using antibody-conjugated magnetic beads, as exemplified by our MACS® MicroBead technology.” Wittmann elaborates that the need for cell enrichment depends on the aim of the experiment. “If you want to sort cells, enriching them can certainly decrease sorting time,” she says. “However, for flow, you could just ‘acquire’ more cells.”
Other suggested staining strategies include the selection of antibodies proven to work in flow cytometry; comparison of existing protocols and literature to identify the most suitable method for a specific experiment; use of directly-conjugated primary antibodies to increase plex and shorten experimental timelines; protection of fluorophores from exposure to light; and the incorporation of automation to improve throughput and reproducibility.
“As spectral imaging and high-plex flow cytometry become increasingly available to researchers, there is a movement away from experiments asking ‘who’ the cells are to what they are doing,” says Manning. “Flow cytometry enables rapid quantification of signaling activity and biological response within each individual cell, while simultaneously providing its identity. This combination continues to provide novel insights, and the field is primed for further innovation.”