Strategies for Intracellular Flow Cytometry Success

Intracellular flow cytometry has seen increased uptake in recent years, driven partly by the growing popularity of immunotherapies, and it is now common practice for cell surface and intracellular markers to be combined in the same flow cytometry experiment. This article comments on the types of intracellular markers analyzed by flow cytometry and provides tips for successful detection.

An evolving approach

Intracellular flow cytometry has been around since the 1970s, when cell cycle studies were performed using acridine orange and intracellular pH was monitored with fluorescein diacetate. Yet, it was not until 1980 that antibody-based detection of intracellular markers was first reported, paving the way for major advances within the field. Now, intracellular staining is used to detect many different analytes. “Typical markers include cytokines, inflammatory mediators, intracellular proteins, nuclear proteins, transcription factors, phosphoproteins, DNA, and fluorescent protein expression,” reports Sharon Sanderson, Senior Application Scientist at Bio-Rad. “In addition, pH, calcium levels, cell cycle progression, cellular proliferation, and metabolic activity though methods such as the DHR assay can also be monitored.”

Revealing novel insights

Critically, using flow cytometry for monitoring intracellular targets can yield insights that might otherwise be overlooked using traditional bulk population analysis methods like western blot or ELISA. “A major strength of flow cytometry is that it allows identification of multiple populations within a single sample,” explains Rob MacDonald, Scientist in the Flow Cytometry Group at Cell Signaling Technology. “Starting from a heterogeneous pool of cells, such as splenocytes or peripheral blood mononuclear cells, researchers can use antibodies against CD markers to identify distinct subpopulations while also staining for intracellular markers. This approach can reveal targets that are differentially expressed, which generally provide more valuable information than widely or ubiquitously expressed markers. For example, monitoring phosphorylated proteins in this way can help determine the activation status of signaling pathways within specific cell subpopulations.”

Consequences of fixation and permeabilization

While detecting cell surface markers on live cells is relatively straightforward, intracellular flow cytometry presents inherent challenges. “Intracellular flow cytometry requires fixation of cells to maintain their structure, followed by permeabilization to allow antibody reagents access to their targets,” says Natalie Oxford, R&D Manager for Cell Biology at Thermo Fisher Scientific. “Fixation may change epitopes, meaning antibodies against surface markers are no longer able to bind, whereas permeabilization can introduce further problems.” These include denaturation of surface-bound protein fluorophores used for cellular identification (e.g., phycoerythrin and allophycocyanin) where organic solvents like ethanol or methanol are employed for permeabilization, and accidental leaching of proteins from cells where Triton™ X-100 is used to provide access to nuclear targets. Sanderson adds that permeabilization can also cause a loss of cell morphology resulting in altered forward (FSC) and side scatter (SSC) profiles compared to intact cells, requiring researchers to adjust PMT voltages for FCS and SSC parameters to ensure they are still on scale.

Best practices for intracellular flow cytometry

Various strategies have been developed to improve researchers’ chances of intracellular flow cytometry success. MacDonald comments that a tried-and-trusted approach is to stain live cells with surface marker antibodies first, before fixing, permeabilizing, and staining for intracellular analytes—remembering to factor in the permeabilization considerations mentioned previously. “Small molecule fluorophores, like fluorescein isothiocyanate and the Alexa Fluor^® dyes, retain their fluorescent properties when carried through a methanol permeabilization step and can be used in place of fluorescent proteins provided they are compatible with the flow cytometer’s lasers and detectors,” he says. “Alternatively, to both circumvent the issue of protein fluorophore denaturation and save time, samples can be fixed and permeabilized prior to staining for surface and intracellular markers simultaneously. Before opting to go down this route, it is important to confirm that surface marker antibodies will still yield accurate results when used on fixed and permeabilized cells; performing a comparative study with known positive and negative samples and using the antibody manufacturer’s recommended staining protocol as a control is a quick and easy way to check this.”

Intracellular staining can often be susceptible to higher non-specific antibody binding and background signal than cell surface staining. Kenta Yamamoto, Product Manager, Cell Analysis at BioLegend, suggests comparing different blocking reagents and increasing the number of wash steps to minimize background staining and titrating antibodies to ensure an optimal signal-to-noise ratio. “Adopting a step-wise approach to optimize your intracellular flow cytometry experiments is key,” he reports.

“This should encompass everything from the method used for sample collection through to data analysis, and it is worth considering using reagents developed specifically for intracellular staining applications.” As an example, he highlights a recent Nature Communications publication, where researchers used BioLegend’s Cyto-Fast™ Fix/Perm Buffer Set for flow cytometry-based analysis of NK cells to determine how these contribute to retroviral immunity. The results of this study have indicated that targeting NK cell mitochondrial fitness may represent an effective therapeutic strategy for retroviral infections.

Controls are another important consideration for intracellular flow cytometry, especially those used to evaluate background staining. “Isotype controls were developed for surface staining and may not be appropriate for assessing background in intracellular protocols,” notes Oxford. “One way to correctly draw gates is to ensure that untreated cells are included as a control whenever staining cells that have undergone any stimulation or other treatment. It is also helpful to have an internal negative control—a population of cells that does not express the target of interest but has undergone all the same treatment and preparation as the positive cells—for intracellular staining experiments to account for any differences in autofluorescence between treated and untreated cells, or between the isotype control and specific antibody.”

Lastly, Sanderson stresses that biological controls should be designed to show the full range of expression of intracellular proteins. “In the case of cytokine detection, a protein transport inhibitor such as Brefeldin A or Monensin can be added to cells for a few hours prior to staining,” she says. “This allows accumulation of proteins that would normally be secreted, enabling their detection. Where cytokines are weakly expressed and/or only expressed by a small percentage of cells, bright fluorophores are preferred, and secondary antibodies can also be used to amplify the signal.”

Six top tips for intracellular flow cytometry

• If you plan to use a surface marker antibody on fixed and permeabilized cells, confirm it still recognizes its target after fixation and that the fluorophore label is compatible with the permeabilization reagent
• Some common viability dyes, such as PI, 7AAD, and DAPI, are not suitable for use in a fixation protocol; try replacing these with a fixable alternative, such as BioLegend’s Zombie Dyes, Bio-Rad’s VivaFix Cell Viability Assays, Cell Signaling Technology’s Ghost Dyes or Thermo Fisher Scientific’s LIVE/DEAD™ Fixable Viability Dyes
• Consider using reagents developed specifically for intracellular staining applications, such as BioLegend’s Cyto-Fast™ Fix/Perm Buffer Set or Cell Signaling Technology’s Intracellular Flow Cytometry Kit
• Review the literature to identify antibodies that have been cited for intracellular flow cytometry, such as Thermo Fisher Scientific’s IL-22 Monoclonal Antibody (22URTI) used for studying gastric cancer progression and Cell Signaling Technology’s Alexa Fluor^® 488 conjugated Phospho-Histone H3 (Ser10) (D2C8) XP^® Rabbit mAb used for investigating cellular bioenergetics during mitosis
• Adopt a step-wise approach to workflow optimization, ensuring conditions enable reliable detection of every target in a multiplexed panel
• Always use suitable controls for validating assay performance and verifying results, including untreated cells, internal negative controls, and positive controls that show the full range of expression of intracellular proteins