Generating accurate flow cytometry data requires more than just specific and sensitive antibodies validated for flow cytometry—it also depends on the right combination of support reagents. When designing your flow experiment, identifying the correct reagents, such as permeabilization buffers, viability dyes, and Fc blockers is important to prevent false positives or negatives and reduce potential cross-reactivity to ensure results you can trust.
We connected with our Associate Director of Flow Cytometry, Christopher Manning, to answer some common flow cytometry troubleshooting questions. Read on for cell preparation tips for intracellular and live cell flow cytometry, or use the links below to jump to a specific topic of interest:
- Live cell vs intracellular flow cytometry vs FACS: What’s the difference?
- How do I fix and permeabilize cells for intracellular flow cytometry?
- How do I confirm cell viability in my sample?
- When should I use Fc block?
- What controls should I include?
Live cell vs intracellular flow cytometry vs FACS: What’s the difference?
Before we dive into cell preparation tips, let’s first level set with a few basic definitions. Although the terms are often used interchangeably, flow cytometry and fluorescence-activated sorting (FACS) are actually different techniques, both of which are used to study complex cell populations. FACS is a subset of flow cytometry used to sort and isolate specific cell populations based on fluorescence signals. While flow cytometry provides analytical data, FACS can also serve as a preparative technique, allowing cells of interest to be physically separated for subsequent experimentation or analysis.
"It's worth noting that while FACS is most commonly performed with live cells, sorting may also be performed on fixed cells for downstream applications such as RNA sequencing," explains Manning.
What about live cell vs intracellular flow cytometry?
- Live Cell Flow Cytometry focuses on the analysis of surface markers on viable cells. This approach is typically used when a researcher needs to identify and distinguish between multiple cell populations within a sample, such as PBMCs, and only needs to stain phenotypic markers expressed on the cell surface to do so. On the CST website, you’ll see this called Flow Cytometry (Live).
- Intracellular Flow Cytometry, on the other hand, involves analyzing proteins within the cell, such as phosphorylated proteins or transcription factors, and, if they're experimentally relevant, cell surface markers can also be identified at the same time. Because the antibodies need access to intracellular targets, cells must first be fixed and permeabilized before analysis. On the CST website, you’ll see this called Flow Cytometry (Fixed/Permeabilized).
Both live cell and intracellular flow cytometry have protocol considerations that can affect the integrity of your results. Especially for intracellular flow, the protocol considerations can sometimes feel daunting, and the potential for acquiring misleading or incorrect data increases. However, if you take some cell preparation considerations into account, generating accurate flow cytometry data for intracellular targets may not be as difficult as you think.
How do I fix and permeabilize cells for intracellular flow cytometry?
When your target is intracellular, proper fixation and permeabilization are essential to prevent false negatives and ensure accurate detection. Fixation stabilizes proteins and preserves their state within the cell, while permeabilization allows antibodies to penetrate membranes and bind to their intracellular targets. If the cell is not completely fixed before the cell membrane is permeabilized, intracellular proteins can leak out of the cell. Conversely, if the cell membrane or relevant organelles are not sufficiently permeabilized, antibodies will not be able to reach and bind to your protein of interest.
The CST® Intracellular Flow Cytometry Kit (Methanol) #13594 contains all the reagents you’ll need to preserve protein states and facilitate efficient antibody access to intracellular proteins. It utilizes methanol-free formaldehyde to fix cells and methanol for permeabilization.
“We often recommend methanol for permeabilization because it’s highly effective at opening up both the plasma membrane and intracellular compartments, giving antibodies access to organelles and other regions throughout the cell,” Manning says. "We’ve seen issues where researchers use less thorough permeabilization methods, leading to poor antibody binding and missed targets. Since methanol does the most thorough job, we typically recommend it as the default method for permeabilization."
Related resource: Flow Cytometry Troubleshooting Guide
However, there are rare cases when methanol is not compatible with the antibody and/or epitope in your experiment, and its use could cause inaccurate results. Not sure if your experiment falls into this category? CST scientists have done the testing for you. When you use a CST antibody, we’ll recommend the appropriate permeabilization protocol on the antibody datasheet or website product page, such as those that use Intracellular Flow Cytometry Kit (Triton X-100) #51995 or FoxP3/Transcription Factor Fixation/Permeabilization Kit #43481, if these methods will yield better results.
Kit components are also available à la carte, providing you with flexibility when designing your experiment.
How do I confirm cell viability in my flow cytometry sample?
Cellular responses, signaling, and protein levels observed in live cells and dead/dying cells can be very different. Including cells in your data analysis that were not viable in your sample at the time of fixation may skew your data. Fixable viability dyes such as the Ghost Dyes offered by CST differentiate between live and dead cells in fixed and permeabilized samples, removing this potential source of bias. They are amine-reactive dyes that covalently bind their target, brightly labeling dead cells prior to fixation and ensuring that you can subsequently fix and permeabilize your sample without losing signal. The signal from the Ghost Dye can then be used to gate non-viable cells and exclude them from your analysis.
“We’ve seen cases where researchers run complete experiments using costly samples, only to realize that viability wasn’t accounted for correctly,” explains Manning. “Viability dyes like Ghost Dyes help you ensure your data aren't skewed by dead cells, which allows you to focus only on biologically meaningful signals.”
Flow cytometric analysis of live and heat-killed mouse bone marrow cells, combined and stained with Ghost Dye Blue 516 Viability Dye #99283. Viable gate is indicated.
CST offers Ghost Dyes in a variety of colors, so you can choose the dye that fits best in your panel of antibody conjugates.
Want to learn more about viability dyes, including dyes that will work for live cells? Check out this blog: Why use viability dyes in flow cytometry experiments?
When should I use Fc block in my flow cytometry experiment?
Fc receptors on the cell surface of immune cells like B lymphocytes, NK cells, macrophages, neutrophils, and mast cells can recognize the Fc fragments of all antibodies, leading to false positives. For example, let’s say you’re using rabbit-derived antibodies to target immune cell markers to characterize immune cells and/or monitor surface proteins of interest in a human sample. The human Fc receptor on your live cells will bind to your rabbit antibodies no matter what your target protein is, causing false positives and potentially making your results less clear-cut. Therefore, Fc blocking should be used anytime you’re performing flow experiments on immune cells with Fc receptors.
“It's not obvious that human Fc receptors will bind to rabbit antibodies,” explains Manning. “What complicates things further is that oftentimes, when a researcher sees an unexpected signal, they assume the antibody is malfunctioning when, in reality, it’s the receptor interaction causing false positives. This makes Fc blocking essential—otherwise, you can’t be certain if your antibody is binding to the right target or just sticking to the receptor.”
Fc Blockers bind to the Fc receptor on your cell before you introduce your primary antibody to the sample, improving your signal-to-noise by ensuring the antibodies are not sequestered by the Fc receptor and are free to bind target proteins.
Flow cytometric analysis of THP-1 cells blocked with Human Fc Receptor Blocking Solution #58948 (2.5 µg/million cells, 10 min; green) or unblocked (blue) and then incubated for 60 min with 5 µg/mL of PE-conjugated isotype controls. Orange dashed line indicates unblocked and unstained cells. Blocking Fc receptors reduces non-specific binding, as shown by the lower fluorescence in the blocked samples.
The Human Fc Receptor Blocking Solution #58948 from CST can be used to prevent an antibody from binding to Fc receptor proteins when performing experiments on live human models that include cells of myeloid/monocyte lineage, while the Mouse Fc Receptor Blocking Solution (CD16/CD32) Rat mAb #88280 can be used to block interaction with mouse Fc receptors. Fc blockers are especially important for live cell experiments, but are generally not needed for intracellular flow cytometry, since fixation and permeabilization disrupt Fc receptors and prevent them from binding antibodies.
What controls should I include in my flow cytometry experiment?
Experimental controls are a must if you want to ensure the validity of your data interpretation. Isotype controls from CST confirm that the signal you see is specific and not due to non-specific binding or artifacts. They can be used when analyzing fixed/permeabilized cells to establish non-specific fluorescence background signals, but are particularly important when using live cells due to the high levels of Fc receptor binding that may occur. The signal from the isotype control ensures positive events are meaningfully quantified and gated relative to background fluorescence levels.
Flow cytometric analysis of Jurkat cells, using Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 647 Conjugate) #2985 (red) compared to Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) #4851, untreated (blue) or IFN-α-treated (green).
Go with the Flow: Data You Trust with Flow Cytometry Application Reagents
CST application scientists are closely involved in developing both the antibodies and companion reagents necessary for flow experiments—and because all our flow-specific reagents have been designed, developed, and tested by application area experts, you can trust that they’ll work in your application, the first time and every time. So you can say goodbye to ambiguous flow cytometry results due to inadequate sample preparation, signal that is not specific to your protein of interest, or the presence of dead cells in your sample.
CST Kits and Reagents for Flow Cytometry
Additional Resources
- For a full list of control and secondary antibodies, buffers, detection, and other reagents to complete your flow cytometry workflow, click the link for Instant access to the Application Workflow Reagents brochure.
