When selecting reagents for flow cytometry, fluorescently-labeled antibodies are likely the first products that come to mind. However, supplementary reagents are also important and it pays to investigate what’s on offer. This article looks at some of the different types of supplementary reagents you should consider for your flow cytometry experiment, with insight from Mike Blundell, Ph.D., Product Manager at Bio-Rad, Eric Torres, Ph.D., Marketing Manager at Biotium, and Christopher Manning, Associate Director, Flow Cytometry at Cell Signaling Technology (CST®).

Cell Viability Dyes

Exclusion of dead cells in flow cytometry is essential to prevent false positives. Dead cells have high autofluorescence and bind to antibodies non-specifically, which can lead to inaccurate interpretation of results. One way of measuring cell viability is to use non-permeant DNA binding dyes, such as propidium iodide (PI), DAPI, or 7-AAD, which can only enter cells with compromised membranes. Biotium’s NucSpot® Nuclear Stains are another option, giving researchers access to a wider selection of colors.

Alternatively, if an experiment requires fixed cells, a fixable cell viability dye should be chosen. “Fixable cell viability dyes typically work by binding free amines on the cell surface, as well as intracellular amines that are exposed in cells with compromised cell membranes, meaning that dead or dying cells having a greater level of fluorescence than their viable counterparts,” explains Blundell. Available options include Bio-Rad’s VivaFix Dyes, Biotium’s Live-or-Dye Fixable Viability Stains, and Ghost Dyes from CST.

VivaFix Cell Viability Assay chemistry. A. Live cells with VivaFix Dye bound to surface primary amines. B. Dead cells with VivaFix Dye bound to surface and intracellular primary amines.

Activation Reagents

Activation reagents are used for purposes that include stimulating intracellular signaling pathways, increasing cytokine production, and driving cell proliferation. The type of activation reagent you choose will depend on the aim of your flow cytometry experiment. “At CST, we offer an extensive selection of Cytokines and Growth Factors, as well as a growing number of Small Molecule Compounds, to meet various cell activation needs,” reports Manning. “In addition, we have developed Rapid-Act T Cell Activation Kits for both human (#88179) and mouse (#86772), which enable easy, single-step induction of T cell activation and proliferation. We also provide a Cell Stimulation Cocktail, either with (#23318) or without (#29255) protein transport inhibitors, to increase cytokine production for detection by downstream immunoassays.”

Rapid-Act T Cell Activation. Flow cytometric analysis of mouse splenocytes, untreated (left column) or treated with Rapid-Act T Cell Activation Kit (Mouse, Anti-CD3/CD28) (15 min; right column), using Phospho-SLP-76 (Ser376) (E3G9U) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #47876 (top row) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 488 Conjugate) #2975 (bottom row), and co-stained with CD3 (17A2) Rat mAb (APC Conjugate) #24265.

Protein Transport Inhibitors

Protein transport inhibitors such as monensin and Brefeldin A are commonly used to enhance intracellular cytokine staining. They achieve this by preventing cytokine secretion, which leads to a rapid accumulation of cytokines in the Golgi apparatus and endoplasmic reticulum. “Both monensin and Brefeldin A are widely available, although the choice and culture conditions for these inhibitors may require optimizing for the species and cytokines being stained,” cautions Blundell.

Red Blood Cell Lysis Buffers

Red blood cell (RBC) lysis is routinely performed when working with whole blood or tissue homogenates in order to properly gate leukocytes. It can involve treating the entire sample prior to staining the remaining cells with fluorescently-labeled antibodies or can be performed once the staining process is complete. In either scenario, it is critical that the lysis buffer has minimal effect on the leukocytes. Commercially available products include Bio-Rad’s Erythrolyse Red Blood Cell Lysing Buffer, which contains a fixative reagent, and CST’s RBC Lysis Buffer(10X) #46232, which is fixative-free.

Cell Cycle Staining Dyes

Cell cycle analysis based on the quantitation of DNA was one of the earliest applications of flow cytometry, and fluorescent dyes that bind stoichiometrically to DNA, such as DAPI, PI, Hoechst 33342, 7-AAD, DRAQ5® and DRAQ7, have all stood the test of time. More recently, alternatives to these products have been developed to increase flexibility for experimental design, including Biotium’s NucSpot® 470 and NucSpot® Far-Red Nuclear Stains. “NucSpot 470 enables selective detection of dead cells by flow cytometry in the FITC channel, while NucSpot Far-Red is an improved alternative to 7-AAD with a red-shifted fluorescence emission for less bleed-through in the PE-Texas Red® channel,” reports Torres.

Cell Proliferation Dyes

Tracking cell proliferation/division is common in flow cytometry. It has traditionally been performed using carboxyfluorescein succinimidyl ester (CFSE), an amine-reactive derivative of fluorescein that is non-fluorescent until it enters viable cells, where it is hydrolyzed by cytoplasmic esterases to yield green fluorescence. However, limitations of CFSE include cell toxicity, leakage from the cell, and bleed-through into the PE and PE-TexasRed® channels.

To address these issues, Biotium has developed ViaFluor® SE Cell Proliferation Dyes. “Our ViaFluor SE Cell Proliferation Dyes improve on many of the properties just described,” says Torres. “They include ViaFluor® 405 SE and ViaFluor® 650 SE, which are respectively detected in the Pacific Blue® and APC channels, and ViaFluor® 488 SE, which can directly replace CFSE for detection in the FITC channel. Not only do ViaFluor Dyes offer better peak separation than CFSE, but they also have less leakage and lower toxicity than CFSE when used at the recommended concentration.”

Principle of cell division tracking with ViaFluor® Cell Proliferation Dyes. When a stained cell divides, each daughter cell receives half the dye in the parent cell, with each cell division represented as a successively dimmer population on a flow cytometry histogram. Data shown using 5 µM ViaFluor® 405 to stain PBMCs.

Bio-Rad’s CytoTrack Cell Proliferation Dyes are another option, supplied in blue, green, and red emission spectra. “CytoTrack Dyes are compatible with standard formaldehyde-containing fixatives and saponin-based permeabilization buffers,” notes Blundell. “Compared to CFSE, they provide clearer resolution of up to ten cell generations.”

Fc Blocking Reagents

Fc receptors on the surface of immune cells such as monocytes, macrophages, and B cells bind antibodies via their constant (Fc) domains, which can lead to false positives. A common way of avoiding this problem is to introduce an Fc blocking step into the staining protocol, such that only antigen specific binding is observed. For this approach to be effective, the Fc blocking reagent should be matched to the sample host species. Available products include Bio-Rad’s Human Seroblock and Mouse Seroblock, and CST’s Human Fc Receptor Blocking Solution #58948 and Mouse Fc Receptor Blocking Solution (CD16/CD32) Rat mAb #88280.

Buffers

Flow cytometry experiments use many different types of buffers, including for fixation, permeabilization, and immunostaining. While many of these can be made in-house, researchers often prefer to use commercial products for the greater consistency they can provide.

“Besides standard flow cytometry buffers, CST offers several reagents that address specific research needs,” comments Manning. “For example, our FluoroClear Blocking Buffer #33449 is a fairly unique product that reduces fluorescent background resulting from weak fluorophore interactions in fixed/permeabilized cells without reducing specific signal resulting from antibody-target interactions. We also offer several flow cytometry fixation/permeabilization kits that enable researchers to exactly replicate the conditions that were used during our extensive antibody validation process. These include our Intracellular Flow Cytometry Kit (Methanol) #13593 and Intracellular Flow Cytometry Kit (Triton X-100) #51995. The inclusion of the permeabilization reagent in the product name is important so researchers can verify proper permeabilization for their targets of interest, and to avoid unintended problems, such as denaturation of protein fluorophores with methanol.”

Supporting Your Research

If you need help with selecting the right dyes to monitor cell viability, perform cell cycle analysis, or track cell proliferation, you can count on FluoroFinder! Use our Fluorescent Dye Database to explore the optical properties and spectral profiles of more than a thousand fluorochromes, and enter your instrument laser and filter combinations into our interactive Spectra Viewer to find the best fluorochrome for your application.

Guest Authored By: Vera Tang, Ph.D.

Interest in the study of extracellular vesicles has increased exponentially over the past two decades due to the important role they play in inter-cellular communication, as biomarkers of disease, and vehicles for drug delivery.

What are Extracellular Vesicles and Why Study Them?

Extracellular Vesicles (EVs) span a large range of sizes from about 20-30 nm to several microns, consisting of many types of particles produced through various biological processes. The generic term of “extracellular vesicles” was adopted in 2014, by the International Society for Extracellular Vesicles (ISEV) as “encompassing all cell-released, membranous particles” in the Minimum Information for Studies of Extracellular Vesicles (MISEV) guidelines¹. In 2018, this definition was appended to include “particles naturally released from the cell that are delimited by a lipid bilayer and cannot replicate”². In 2023, the word “naturally” was removed from the definition³. Terms identifying different types of vesicles such as exosomes, microvesicles, and apoptotic bodies, were more commonly used prior to the adoption of the generic term of EV. These terms are still used when the specific biogenesis mechanisms of the particles of interest are explicitly demonstrated. Size alone does not identify the type of EV. It is also worth noting that, in certain interpretations, the definition of EV could potentially include some viruses.

Why are EVs Challenging to Study?

Simply put, EVs are very small. This makes it challenging to detect them using commonly available lab equipment such as flow cytometers and light microscopes because most EVs are at or below the limit of detection of many of these technologies. The small size of these particles inherently means there is limited surface area to express proteins (e.g. CD markers) and internal volume to hold relevant cargo (e.g. mRNA); common targets for EV phenotypic studies. EVs can be expected to have very few surface proteins and internal cargo for detection in comparison to cells. Theoretically, a 10 µm cell is 100 times larger than a 100 nm EV in diameter but has 10,000 times the surface area and 1,000,000 times the volume. To give an example, a typical human CD4 T cell expresses approximately , 50,000 to 150,000 molecules of CD4 on its surface^4,5. If we assume the antigen density of CD4 is the same on the EVs as that of the T cells producing them, the number of CD4 molecules on an EV that is 100 times smaller than the cell (in diameter) will be in the order of 5-15 molecules.

EV samples can be heterogeneous depending on the source. EVs isolated from liquid biopsies, such as blood and urine, may contain many different types of EVs as well as proteins and endogenous viruses. Viruses and virus-like particles can also share many of the same markers used to identify EVs⁶ and similar structural components and physical characteristics such as lipid membranes and buoyant densities⁷. In contrast, EVs isolated from cell lines in vitro are more homogenous as they are derived from a single cell type. Generally, phenotypic characterization of EVs benefits from utilizing single-particle detection technologies and not bulk analyses because biological variation exists even in the simplest of samples. Single-particle detection technologies, such as flow cytometry, analyze individual particles in the sample. Bulk analysis techniques, such as western blotting, provide an average measurement of all the particles combined, masking any heterogeneity within a sample. Nonetheless, western blotting consistently remains the most used method for analysis of EVs in world-wide surveys conducted by ISEV with only ~50% of respondents choosing flow cytometry as a technique utilized in their lab^8,9.

Flow Cytometry as a Tool to Study EVs

Flow cytometry is a technology designed to provide information on both the fluorescence and size of individual particles, namely cells, with a high degree of sensitivity.  The high-throughput and single-particle basis of detection has made flow cytometry the go-to method for characterizing heterogeneous cellular populations with numerous parameters spanning multiple decades in fluorescence intensity signals. Nevertheless, EVs can be challenging to analyze, requiring additional controls and alternate approaches to analysis than those used for cells.

Most Flow Cytometers are Designed to Analyze Cells, Not EVs

Coincidence

The sample core stream of most flow cytometers will be much wider than the diameter of EVs. This significantly increases the occurrence for coincidence measurements, which is the simultaneous detection of multiple particles as a single event. Specific controls and procedures are recommended in the Minimum Information about a Flow Cytometry experiment on EVs (MIFlowCyt-EV) reporting framework to verify that single particle measurements are being made¹⁰.

Settings Optimization

The detection of EVs requires the use of flow cytometers at the limit of detection. This means that the standard settings used for detection of cells will not adequately resolve EVs and other small particles.  The manufacturer’s instrument quality control software to monitor performance was not designed with the intention of using the instruments at the limit of detection (LoD). There is yet to be a consensus method to detect EVs or to optimize instrument settings for small particle detection, although a workflow was proposed by Cook et al. in 2023¹¹, detailing a method for instrument detector settings optimization and data calibration for standardized reporting.

Standardization of Data Reporting

Flow cytometers collect data in the form of light intensity. These measurements are arbitrary and lack standard units for direct comparisons between different instruments and timepoints. To make quantitative comparisons, flow cytometry data requires the use of controls, calibration materials and software. Methods to calibrate fluorescence¹² and light scatter¹³ data from cell samples into standard units have existed since the 1980’s. These methods have been demonstrated in small particle samples in the recent two decades^14–16.

Data reporting in standard units is especially important for analysis of EVs and other small particles because these particles of interest are at or below the instrument limit of detection. Importantly, this limit of detection will also differ between instruments, irrespective of manufacturer and model. In a recent cross-platform comparative study with four Cytoflex S and four Aurora ESP analyzers utilizing the same method of settings optimization, the LODs for these flow cytometers ranged from 29 to 155 EGFP MESF and light scatter sensitivity from 72 to 100 nm for detection of a recombinant GFP+EV reference sample^11ix.  Other studies comparing a wider range of flow cytometers have reported even greater variability in detection sensitivity for light scatter^15,17. Differences in LoD directly impact the number of EVs detected in an exponential manner^18,19 and have implications on the statistics that should be reported for EV flow data.

The percentage of cells positive for specific markers is a commonly reported statistic in flow cytometry. Unfortunately, this same statistic does not hold similar merit in EV flow cytometry data because, as discussed above, not all EVs are resolved and the amount of EVs detected varies with the LoD of the instrument. Reporting the concentration of EVs expressing a marker of interest within a certain size and intensity of expression (in standard units) is a reproducible way to report statistics associated with EVs outlined in the MIFlowCyt-EV Reporting Framework¹⁰.

Reagents

Most commercially available flow cytometry reagents are designed for applications in cellular detection assay and are not validated for use in EVs. In the case of fluorescent dyes and conjugated antibodies, aggregates of these reagents can be misidentified as labeled EVs if the appropriate controls are not used. In many cases, washing of EV samples by centrifugation is not feasible and so titration of reagents becomes crucial to optimization of sample labeling from background. Many commercial fluorescently conjugated antibodies are sold in “tests” and do not readily disclose the concentration. This makes it very challenging to standardize and compare between reagent lots.

In consideration of these many challenges to analyze EVs by flow cytometry, the small particle/EV flow cytometry community has produced numerous educational resources and frameworks to support researchers in these studies. For those new to the field, the following provide some important background information to get you started:

• A compendium of single extracellular vesicle flow cytometry²⁰
• MIFlowCyt-EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments¹⁰
• The most recent MISEV update³

What’s Next on the Horizon?

Hardware

We are seeing an increase in commercially available flow cytometers that are dedicated to small particle detection and while this may not be for every budget, add-ons for more sensitive light-scatter detection are now available on analyzers from most major flow cytometry vendors. Dedicated small particle flow cytometers are now capable of resolving 40 nm polystyrene beads from noise by light scatter (CytoFLEX Nano, NanoFCM). Increasing sensitivity in fluorescence detection for single molecule resolution will be the new frontier as this will be required for the phenotypic characterization of the smallest detectable EVs.

Software

As the analysis of EVs and other small particles like viruses become more common, instrument manufacturers need to incorporate more detailed quality control (QC) measures to monitor performance as well as instrument service to support these types of assays. Especially with respect to dedicated small particle flow cytometers, incorporating well characterized reference materials into more detailed QC procedures will give more quantitative metrics to monitor instrument performance. Software for data calibration such as FCMPASS²¹ and Rosetta Calibration²² are currently separate from acquisition. Beckam Coulter currently provides FCMPASS software with the purchase of the CytoFLEX Nano²³ and Rosetta Calibration is available as a plug-in for FlowJo²⁴. However, to facilitate wider adoption of standardized data reporting, calibration should be incorporated in the acquisition software such that raw data from a flow cytometer can be exported in standard units.

Reagents

With respect to antibodies, having more information on specific antibody lots including the concentration and fluorophore to protein ratios and better removal of aggregates would be beneficial for both cell and EV based assays. Vendors such as Biolegend have taken the lead and implemented a searchable database of their Certificates of Analysis by lot number²⁵. In terms of reference materials for calibration, more high quality, well characterized materials are needed such as the 3000-series Nanosphere Size Standards²⁶ from ThermoFisher which are NIST-traceable and come with a certificate of analysis. A similar need exists for well characterized materials for fluorescence calibration that can work across different detection platforms.

Supporting Your Research

We are at an exciting time for flow cytometry with spectral, imaging, and an ever-growing list of new technologies with improved sensitivity coming to the forefront. Perhaps in the not-so-distant future, the analysis of EVs and other nanometer-sized particles by flow cytometry will be standard procedure and automated software workflows with facilitate reporting in standard units making this a reality for both cell and EV data alike.

For researchers wanting to study EVs using flow cytometry, FluoroFinder has tools designed to support both beginners and experts. Our platform includes thousands of validated reagents. Use the Spectra Viewer to help you select fluorochromes compatible with your instrument’s sensitivity and filters—essential for working at the limits of detection. FluoroFinder also connects you with reference materials, calibration tools, and educational resources that align with MIFlowCyt-EV and MISEV guidelines, empowering you to design reproducible experiments and confidently report results in standardized units. Whether you’re optimizing for rare marker detection or comparing cross-platform data, FluoroFinder helps you stay ahead in this rapidly evolving field.

References:

1. Lötvall, J. et al. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J. Extracell. Vesicles 3, 26913 (2014).
2. Théry, C. et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles 7, 1535750 (2018).
3. Welsh, J. A. et al. Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. J. Extracell. Vesicles 13, e12404 (2024).
4. Wang, L. et al. Variables in the quantification of CD4 in normals and hairy cell leukemia patients. Cytometry B Clin. Cytom. 80B, 51–56 (2011).
5. Wang, M. et al. Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry. Clin. Proteomics 11, 43 (2014).
6. Burnie, J. et al. Identification of CD38, CD97, and CD278 on the HIV surface using a novel flow virometry screening assay. Sci. Rep. 13, 23025 (2023).
7. Nolte-‘t Hoen, E., Cremer, T., Gallo, R. C. & Margolis, L. B. Extracellular vesicles and viruses: Are they close relatives? Proc. Natl. Acad. Sci. 113, 9155–9161 (2016).
8. Gardiner, C. et al. Techniques used for the isolation and characterization of extracellular vesicles: results of a worldwide survey. J. Extracell. Vesicles 5, 32945 (2016).
9. Royo, F., Théry, C., Falcón-Pérez, J. M., Nieuwland, R. & Witwer, K. W. Methods for Separation and Characterization of Extracellular Vesicles: Results of a Worldwide Survey Performed by the ISEV Rigor and Standardization Subcommittee. Cells 9, 1955 (2020).
10. Royo, F., Théry, C., Falcón-Pérez, J. M., Nieuwland, R. & Witwer, K. W. Methods for Separation and Characterization of Extracellular Vesicles: Results of a Worldwide Survey Performed by the ISEV Rigor and Standardization Subcommittee. Cells 9, 1955 (2020).
11. Cook, S., Tang, V. A., Lannigan, J., Jones, J. C. & Welsh, J. A. Quantitative flow cytometry enables end-to-end optimization of cross-platform extracellular vesicle studies. Cell Rep. Methods 3, 100664 (2023).
12. Brown, M. C., Hoffman, R. A. & Kirchanski, S. J. Controls for Flow Cytometers in Hematology and Cellular Immunology. Ann. N. Y. Acad. Sci. 468, 93–103 (1986).
13. Doornbos, R. M. P. et al. Lissajous‐like patterns in scatter plots of calibration beads. Cytometry 16, 236–242 (1994).
14. Welsh, J. A., Jones, J. C. & Tang, V. A. Fluorescence and Light Scatter Calibration Allow Comparisons of Small Particle Data in Standard Units across Different Flow Cytometry Platforms and Detector Settings. Cytometry A 97, 592–601 (2020).
15. Van Der Pol, E. et al. Standardization of extracellular vesicle measurements by flow cytometry through vesicle diameter approximation. J. Thromb. Haemost. 16, 1236–1245 (2018).
16. Stoner, S. A. et al. High sensitivity flow cytometry of membrane vesicles. Cytometry A 89, 196–206 (2016).
17. Standardisation of concentration measurements of extracellular vesicles for medical diagnoses – METVES II. https://www.metves.eu/.
18. Gasecka, A., Böing, A. N., Filipiak, K. J. & Nieuwland, R. Platelet extracellular vesicles as biomarkers for arterial thrombosis. Platelets 28, 228–234 (2017).
19. Cimorelli, M., Nieuwland, R., Varga, Z. & Van Der Pol, E. Standardized procedure to measure the size distribution of extracellular vesicles together with other particles in biofluids with microfluidic resistive pulse sensing. PLOS ONE 16, e0249603 (2021).
20. Welsh, J. A. et al. A compendium of single extracellular vesicle flow cytometry. J. Extracell. Vesicles 12, e12299 (2023).
21. FCM_PASS. https://www.fcmpass.com/.
22. Rosetta Calibration – Exometry. https://www.exometry.com/products/rosetta-calibration/.
23. Optimizing EV Analysis with a CytoFLEX nano flow cytometer and FCMPASS. https://www.beckman.com/resources/reading-material/application-notes/optimizing-ev-analysis-with-a-cytoflex-nano-flow-cytometer-and-fcmpass.
24. Rosetta Calibration. FlowJo Documentation https://docs.flowjo.com/flowjo/plugins-2/plugin-demonstration-videos/rosetta-calibration/.
25. Certificate of Analysis. https://www.biolegend.com/de-de/certificate-of-analysis.
26. 3000 Series Nanosphere^TM Size Standards. https://www.thermofisher.com/order/catalog/product/3020A.

So many new fluorophores have been released in recent years, it can be hard to keep track of their similarities or novelty. Sometimes, a company will create a new brand name for a fluorescent chemistry that is already commonly used, which can make sifting through the relevance of new products a challenge. Let’s break them down by type of chemistry to group fluorophore families with similar characteristics, along with the companies and brand names that fall into these different categories.

Organic Fluorophores

Simple organic fluorophores are the building blocks of all fluorescence found in nature from aromatic amino acids like tryptophan and tyrosine to metabolic products like NADH and FAD to both soluble and insoluble antioxidants like riboflavin and vitamin E (Tocopherol). Common structure of all organic fluorophores is a rigid, conjugated polyaromatic hydrocarbon structure that in its microenvironment, whether an aqueous solution, a lipid membrane or an acidic organelle, it is capable of absorbing energy of a specific wavelength, jumping to an excited state where it can resonate that energy efficiently before emitting it as a photon of light.

These molecules are small, typically less than 2kD and they can be modified to be pH and solvent stable and resist ROS-mediated oxidative degradation like photobleaching. Finally, when they are conjugated to an antibody or other biomolecule, the ratio of fluorophore to protein will reflect the size of both the biomolecule and the fluorophore but is generally 3-6 fluorophore per antibody. This includes their use as the acceptor fluorophore in a FRET tandem with PerCP, PE or APC. Every chemistry serves a special function in an assay and although each has strengths and weaknesses, sometimes even a weakness can be a strength. For example, this family is small and therefore has limited brightness, but this weakness can be used advantageously for fixable viability reagents.

Examples of brand names associated with this fluorophore family includes:

Brand Names			Vendor
Cyanine, FITC, Coumarin, TAMRA, ROX, Rhodamine			Off-Trademark
Brand Names	Vendor	Brand Names	Vendor
iFluor®, XFD	AAT-Bioquest	cFluor*, Ghost	Cytek
HiLyte	Anaspec	DyLight, DY	Dyomics
ATTO	ATTO-Tec	Vio®, Vio®Bright, Viobility	Miltenyi
V450/V500, FVS	BD Biosciences	Janelia Fluor®	Promega
ViaKrome, Krome	Beckman Coulter	CoraFluor	R&D Systems
Spark, Zombie	Biolegend	Seta, SeTau	SETA BioMedicals
CF®, Live-or-Dye	Biotium	eFluor, LIVE/DEAD, Alexa Fluor, Pacific Blue, Bodipy	Thermo Fisher

*Indicates exceptions to this brand

Chromoproteins

Protein-based fluorophores, also called chromoproteins, are naturally produced by algae, cyanobacteria, jellyfish, sea anemones, slugs and corals in the form of fluorescent proteins (FP), R-phycoerythrin (R-PE) and allophycocyanin (APC). The majority of the mass of these structures is a non-fluorescent protein scaffolding which embed small chemical chromophores. When a protein is properly folded, these chromophores become fluorescent, also called fluorogenic, due to the structural rigidity created by the protein microenvironment. Thus, the fluorescence of proteins will be very sensitive to anything that can denature, degrade or digest the protein, like any alcohol in a fix and perm buffer.

Fluorescent proteins (FPs) can be monomeric or polymerize together into dimers, etc. Polymeric FPs can be a hazard when using them for protein localization, in the event they are binding to each other. However, polymeric FPs can also be brighter, shift spectrally to a longer wavelength emission and be useful as a functional probe due to spectral changes that can happen upon aggregation.

R-phycoerythrin (R-PE), peridinin-chlorophyll complex (PerCP) and allophycocyanin (APC) are all commonly used reagents in flow cytometry. PE and APC are very large macromolecules with multiple chromophores generally called phycobilins embedded on different subunits, for example PE is 240kD with 24-30 embedded phycobilins and APC is 105kD with 6 phycocyanobilins. PerCP is 30kD with 8 perdinin complexed to 2 chlorophyl molecules and is much dimmer than either PE or APC and much more photo labile. PE and APC make excellent donor molecules coupled to simple organic dyes in a FRET tandem and with the exception of the Cytek, these producs will be named first as PerCP, PE or APC and then the brand name of the simple organic fluorophore to which it is coupled.

Inorganic Nanocrystals

To increase the number of fluorescent parameters flow cytometry, inorganic nanocrystals were introduced as antibody conjugates around 2005. Generally, inorganic nanocrystals will consist of a metallic core like Cadmium Selenide or Cadmium Telluride encased by a thin coating like Zinc Sulfide to seal the core, a technology which won the Nobel Prize in Chemistry 2023. Others may be composed of colloidal aggregations of metal particles. However, a nanocrystal needs to be functionalized to covalently conjugate a biomolecule or antibody, which is a challenge for a metallic particle. Typically, the particle will be coated in either a spray or “net” of lipids or an amphiphilic polymer to ensure even surface coverage.

The overall size of the particle and the hydrophobic surface can cause some issues with intracellular penetration and reagent aggregation respectively. Despite these challenges, nanoparticles have some unique beneficial properties. Because they are inorganic, they do not succumb to photobleaching like organic fluorophores. Their core can be oxidized under intense direct laser penetration, but not nearly at the same rate as the aforementioned simple organic and protein-based fluorophores. These nanocrystals are often used in solar technologies due to their efficient absorption/excitation at the shortest wavelengths, like UV and 405nm lasers. And finally, they can exhibit tight emission profiles with less spectral spillover into neighboring channels.

ThermoFisher offers QDot nanocrystals and CoreQuantum Technologies offers MultiDots within this family. Also of interest are CoreQuantum’s MagDots which incorporate superparamagnetic iron oxide nanoparticles called SPIONs that enables both fluorescent detection and cell separation. More information can be found on applications for MultiDots and MagDots in our on-demand webinar library.

Multimers and Polymers

Organic multimers and polymers were developed to replace and expand QDot nanocrystal applications in flow cytometry. The Horizon Brilliant fluorescent polymers from BD Biosciences, the SuperNova dyes from Beckman Coulter, the SuperBright polymers from Thermo Fisher, and some of the VioBright Dyes from Miltenyi are of similar structure and utility off the UV and 405nm laser lines in flow cytometry.

Although they are smaller, Brilliant Violet 421 and Brilliant Ultraviolet 395 have similar extinction coefficients (EC) and brightness to PE and APC respectively. High EC values make them excellent donor molecules in a FRET tandem, creating an array of spectrally unique emission profiles. Because they are synthetic organic chemical structures with no protein component, they are also very stable to temperature, solvent and fixatives. However, this chemistry is known to have significant unwanted non-specific binding characteristics, specifically to itself, and a staining buffer is required to prevent this.

From Bio-Rad, an alternative polymer dot called StarBright reagents, overcomes some of these non-specific binding issues and even more importantly offers polymer dots, or pdots, capable of excitation off the 488nm, 561nm and 633nm lasers.

Another advancement in multimer dye technology is the RealBlue and RealYellow fluorophores from BD Biosciences. RealBlue and RealYellow fluorophores are smaller than the traditional polymer chemistry with better intracellular and intranuclear permeability, and a very high brightness.

As the competition grows around this chemistry, we are sure to see even more improvement in the quality, stability and reliability of multimers and polymers.

Supporting Your Research

Our previous article on the Expansion of Fluorophores for Spectral Flow Cytometry may be of interest to you for additional reading on fluorophores. This resource also provides information on DNA backbone polymers that were not described here.

You can use the Dye Directory to search our comprehensive list of fluorophores with spectra and photophysical characteristics and a view of spectrally similar fluorophore alternatives for comparison.

Conjugating antibodies to other molecules is a common technique used for drug delivery, changing solubility, attaching to solid substrates (for purification or assays), and visualizing the antibody using fluorescent, magnetic or gold nanoparticles. Western blots, fluorescence microscopy and flow cytometry all depend on fluorescently tagged antibodies for visualization. Although fluorescently conjugated  antibodies have been available for decades, it’s not always possible to find an off-the-shelf solution for your antigen of interest, so researchers often perform their own conjugation. Conjugating primary antibodies lets the researcher use the same isotype for different antigens in a single protocol- impossible when using labeled secondary antibodies.

Two main approaches for antibody labeling will be discussed in this article. The first involves direct bond formation between reactive amino acid sidechains and a modified fluorophore. This can yield high degrees of labeling (DOL > 5 fluorophores per antibody), but tends to have less specificity. It is important to characterize (DOL, antibody binding properties) and purify the resulting conjugates to verify performance. The second method involves specific labeling of the antibody’s Fc (constant) region with lower DOL (< 5 fluorophores per antibody). Kits are available to simplify both of these procedures, reduce purification steps and provide consistent results. Many companies also provide conjugation services, including Jackson ImmunoResearch Labs, BioLegend, Cell Signal, Fortis and others.

Amino Acid Labeling

Many amino acid side chains contain reactive groups such as thiol/ sulfhydryl, alcohol, carboxylic acids and amines. While there are techniques to conjugate to many of these (Kjaersgaard 2022), the simplest to conjugate are primary amines, the N-terminus of the protein and Lysine (Lys), and Cysteine (Cys), a thiol or sulfhydryl).

Lysine and Primary Amines

Lys is a very common amino acid (in the top 1/3 of abundance) with a primary amine sidechain. It has a pKa of ~10.5 and is mostly positively charged (protonated) at neutral pH. As such, it is preferentially positioned on the outside of a folded protein in aqueous solution, as is the protein’s N-terminus (Jacob 2007) which exhibits similar reactivity. The deprotonated form is one of the most nucleophilic amino acid sidechains. Conjugation reactions proceed by nucleophilic attack of the neutral primary amine of Lys on the carbon atom of isothiocyanate (NCS) or N-hydroxysuccinimide (NHS) ester (Figure 1). For NCS, this yields a substituted thiourea (Figure 1a), and for NHS, it yields an amide bond and the NHS leaving group (Figure 1b).

Figure 1. Lysine conjugation chemistry. The protein’s peptide backbone is shown at the top. F denotes a fluorophore. (A) the nitrogen lone pair attacks the carbon of the isothiocyanate to yield a thiourea (B) the nitrogen lone pair attacks the carbonyl carbon of the NHS ester to yield an amide.

Fluorescein isothiocyanate (FITC), the best-known NCS reagent, has been available since the early 1960s as fluorescence labelling techniques were starting to become mainstream. Other fluorophores available with this reactive sidechain include Rhodamine B, Texas Red and several cyanine dyes. The Lys-NCS antibody conjugation reaction occurs at pH 9-10 over several hours (Figure 1a).

At elevated temperatures, the FITC-conjugated antibody is unstable (Banks 1995), which led to the development of NHS esters in the early 1980s. NHS-ester-fluorophores and kits are available from a large number of manufacturers with a wide array of fluorophores, from the UV and visible through the NIR (for small animal labeling). This conjugation reaction is performed at a milder pH (7-9) and is significantly faster than the NCS reaction, which is why it has largely taken over as the preferred amine-conjugation protocol. The pH dependence of the reaction can be exploited to selectively label the N-terminus (pKa ~ 8.9) at pH 6.5 (Selo 1996). To address the low solubility of NHS-dye complexes, negatively charged sulfo-NHS-dye complexes were developed. The reaction proceeds the same way with both forms of NHS.

Lysine-labelling is effective but there are several things to keep in mind:

• The specificity of this reaction is low, so antibodies should be purified before the reaction. Buffers must be free of other agents that can cross-react, including amines, azides, glycerol, BSA, etc.
• The results are heterogeneous – there is little to no control over the number or locations of labelled Lys residues.
• Fluorescent NCS and NHS reagents have poor aqueous stability. They are typically stored as powders or in dry DMSO/DMF at low temperatures to minimize hydrolysis. The powder or solution should be warmed to room temperature before opening to reduce water condensation.
• Cleanup steps are required to separate labeled antibodies from free dye. These steps can cause loss of precious labeled conjugates – impractical for very small reactions.

Cysteine (Cys)

Free Cys, much less common than Lys (in bottom 1/3 by count) and often covalently bound in disulfide bonds, is the most nucleophilic amino acid when deprotonated, with a pKa around 8.4. It reacts to form relatively unstable conjugates with both the NCS and NHS-esters described above. To increase the stability of the conjugates, maleimide chemistry was developed as an alternative, using a number of maleimide derivatives (Christie 2015).

This coupling proceeds through a Michael addition reaction, as shown in Figure 2. The second hydrolysis step provides stability to the resulting conjugate (Tumey 2014). Unlike Lys, which exhibits similar reactivity regardless of position, the reactivity of Cys with maleimide fluorophores is position-dependent and can be directed by adjusting the reaction conditions (Boll 2022).

Figure 2. Fluorescent maleimide reaction with Cys. The protein’s peptide backbone is shown at the top. F denotes a fluorophore. The first reaction is a Michael addition followed by a ring-opening hydrolysis step.

Like the Lys-reactions, Cys-coupling reactions need to be cleaned up to remove unreacted reagents when the reactions are complete. A number of manufacturers offer fluorescent maleimide reagents, including Biotium, Lumiprobe, and ThermoFisher.

No-Cleanup Covalent Conjugation Kits

New single-reaction sized covalent labeling kits have been developed by Biotium and Abcam to eliminate the post-conjugation cleanup steps. The labels in these kits are stored and shipped in a solid form designed to react with a predetermined quantity of antibody in a single reaction.

Mix-n-Stain^TM kits from Biotium have been uniquely formulated to tolerate low concentrations of amines (<20mM Tris) and other interfering compounds. Reaction buffer is added to the antibody, the solution is transferred to the vial containing lyophilized fluorophore, labeling is completed in 15 minutes in the dark, then a storage buffer is added.  A concentration and purification step can be performed before or after labelling if required. Kits are available for both IgG and IgM isotypes with over 30 CF dye options.

The Lightning Link kit (Abcam) 3-step conjugation kit is comprised of a buffer, lyophilized label mixture and quencher. The concentration of the lyophilized label mixture has been optimized so that there are only low levels of free label remaining at the end of the reaction. Any unreacted, unhydrolyzed label is quenched with a small water-soluble primary amine and washed out during wash steps of the subsequent staining protocol. This kit provides fast, reproducible conjugation. AbCam also sells purification kits to remove albumin, BSA and other confounding proteins from the antibody solution before using this kit.

Fc Specific Labeling

This method labels the Fc (constant) region of the antibody (Figure 3) to eliminate interference of conjugated amino acids with antigen binding in the Fab regions. These methods are not sensitive to buffer type or the presence of other proteins in solution. While they are more specific, the DOL tends to be lower (but less variable) using these methods.

Figure 3. Antibody illustration with Fc region noted.

Over 20 years ago, Molecular Probes (now ThermoFisher) developed Zenon technology, which uses fluorescently labeled antibody fragments against the Fc region of IgGs. Conceptually, this is similar to using fluorescent secondary antibodies. The reagents are specific to the organism (mouse, rabbit, human, goat) and are available with a number of fluorophore tags. At elevated temperatures, the conjugate is unstable which led to the development of newer approaches.

Proteintech’s FlexAble product labels the Fc region of the antibody with a fluorescently labeled polypeptide (FlexLinker) to bind the isotype-specific Fc region of select species (rabbit, mouse, rat, human) in minutes with high affinity. Excess FlexLinker is reacted with FlexQuencher (an IgG or Fc fragment) and washed away during wash steps of the staining protocol. FlexAble conjugation results in 2 fluorophores per antibody, Flexable 2.0 results in 5 per antibody. Both are available with a number of CoraLite and CoraLite Plus dyes.

The oYo-Link method, by AlphaThera, utilizes a genetically engineered protein derived from the Fc binding domain of staphylococcal protein A. The protein contains photoactive benzoylphenylalanine in the binding site, which crosslinks with the antibody backbone upon irradiation at 365 nm, forming a permanent covalent linkage at a designated position. This has been verified in antibodies from a number of species. The oYo-Link reagents are available with allophycocyanin and phycoerythrin fluorescent probes. Up to 2 fluorophores can be bound to the antibody with this method.

Supporting Your Research

There are many strategies for antibody conjugation available to the modern biologist. For high DOL requirements, amino acid conjugation reagents and single reaction kits are available. For more specific binding, with lower DOL and no change to Fab binding regions, both affinity and covalent Fc conjugation options are available.

Whether you’re using off-the-shelf conjugates or planning custom labeling strategies, FluoroFinder helps you streamline your workflow so you can focus on what matters most – your research. Use our Antibody Search to find fluorophores and dyes, or unconjugated antibodies with conjugation kit options. Put it together with our Spectra Viewer to visualize spectral overlap and optimize your marker-fluorophore combinations. 

References:

1. Banks, P.R. and Paquette, D.M. “Comparison of three common amine reactive fluorescent probes used for conjugation to biomolecules by capillary zone electrophoresis.” Bioconjugate Chem 6, no. 4 (July 1995): 447-458.
2. Boll, L.B. and Raines, R.T. “Context-dependence of the reactivity of cysteine and lysine residues.” CHembiochem 23, no. 14 (2022): e202200258.
3. Christie, R.J., et al. “Stabilization of cysteine-linked antibody drug conjugates with N-aryl maleimides.” J. Controlled Release 220 (2015): 660-670.
4. Jacob, E. and Unger, R. “A tale of two tails: why are terminal residues of proteins exposed?” Bioinformatics 23, no. 2 (Jan 2007): e225-e230.
5. Kjaersgaard, N.L., et al. “Chemical conjugation to less targeted proteinogenic amino acids.” ChemBioChem 23, no. 19 (July 2022): e202200245.
6. Selo, I., et al. Preferential labeling of alpha-amino N-terminal groups in peptides by biotin: application to the detection of specific anti-peptide antibodies by enzyme immunoassays.” J. Immunol Methods 199, no. 2 (Dec 1996): 127-38.
7. Tumey, L.N., et al. “Mild method for succinimide hydrolysis on ADCs: Impact on ADC potency, stability, exposure and efficacy.” Bioconjugate Chemistry 25, no. 10 (2014): 1871-1880.

Flow cytometry assays are integral to cell manufacturing, where they have an essential role in safeguarding product quality and function. However, the types of controls used for flow cytometric characterization are changing as researchers realize the advantages of cell mimics over traditional fluorescent beads or biologically-derived materials. We spoke with Annalise Barnette, Ph.D., Senior Manager, Global Marketing at Slingshot Biosciences, who explained how precision-engineered cell mimics serve as more reliable controls for modern cellular testing.

What is Cell Manufacturing?

The term cell manufacturing is generally used to refer to the production of cell therapies – advanced cell preparations that are transplanted into a patient for medical purposes. The cells can either be autologous, meaning they are derived from the individual in question, or allogeneic, meaning they are sourced from a donor. In either scenario, some form of modification is typically required for the cells to perform their intended function. The following are some leading examples of cell therapies:

Chimeric Antigen Receptor (CAR) T cell Therapy

CAR T-cell therapy is a type of immunotherapy that harnesses T cells for targeted tumor cell killing. It involves isolating the T cells from the blood and engineering them to express a CAR at the cell surface; the modified cells are then expanded in culture and infused into the patient. Each CAR has an extracellular antigen-binding domain, which is derived from a tumor-specific antibody single chain variable fragment (scFv), and an intracellular signaling domain, which promotes T cell activation in response to target binding. The US Food and Drug Administration (FDA) has approved six CAR T-cell therapies to date, four targeting CD19 to treat relapsed or refractory B cell cancers and two targeting the B cell maturation antigen (BCMA) to treat multiple myeloma¹.

Stem Cell Therapy

Stem cells have the unique abilities to self-renew and differentiate into other cell types. As a result, they are being investigated for many different regenerative medicine applications, as well as for the treatment of autoimmune disease. Recent publications describe testing of human embryonic stem cell-derived retinal pigment cells to treat age-related macular degeneration; evaluation of human umbilical cord-derived mesenchymal stem cells to alleviate the symptoms of osteoarthritis; and assessment of intestinal epithelial stem cells to suppress inflammatory bowel disease^2,3,4. Currently, the only stem cell products that are FDA-approved consist of hematopoietic progenitor cells that are derived from umbilical cord blood⁵.

Tumor-Infiltrating Lymphocyte (TIL) Therapy

TIL therapy involves isolating the natural infiltrating lymphocytes from a tumor, expanding them in vitro, and infusing them back into the patient with a high dose of interleukin-2 to stimulate TIL activity. Because TILs are composed of multiple clones, they have the potential to address challenges posed by tumor heterogeneity⁶. However, following FDA approval of the first TIL therapy in 2024 (lifileucel for adult patients with advanced or unresectable melanoma), the supporting clinical trials have identified possible adverse events that highlight the need for further development⁷.

The Importance of Flow Cytometry to Cell Manufacturing

Flow cytometry represents an essential technique for cell manufacturing, where it is used to monitor critical quality attributes (CQAs) throughout the entire product lifecycle. Applications of flow cytometry include confirming cellular phenotype and function, checking for contaminating cell types, and monitoring the surface expression of CARs. However, because flow cytometry currently lacks international reference standards, product developers must ensure that flow cytometry experiments are tightly controlled in order to generate reproducible and accurate data.

How do Cell Mimics Improve on Standard Controls for Flow Cytometry?

Standard controls for flow cytometry consist of either fluorescent beads or biologically-derived materials. A major advantage of fluorescent beads is that they represent a convenient, shelf-stable solution. However, this benefit is offset by the fact that fluorescent beads can never fully resemble biological samples. Live cells are more biologically relevant controls, comprising hugely complex systems. Yet live cells face stability, consistency, storage, and supply-chain issues, amongst other challenges. To overcome the various limitations of standard controls, there is an increasing drive towards the use of alternative solutions, such as cell mimics⁸. Not only are cell mimics more consistent than traditional controls, but they also have superior stability, are non-biohazardous and easily scalable, and allow for customization.

Tunable Cell Mimics from Slingshot Biosciences

Slingshot Biosciences’ cell mimics* allow for tuning of cellular characteristics such as granularity, size, refractive index (RI), surface biomarker expression, and fluorescence. In addition, it is possible to embed DNA or RNA in cell mimics and create controls for orthogonal validation of sequencing assays.

“Besides being highly consistent, our cell mimics can be manufactured at scale without sacrificing performance or reliability,” reports Barnette. “They are also incredibly easy to use – simply reconstitute the lyophilized product in PBS, perform any necessary staining, and run the flow cytometry assay as per usual.” Figure 1 shows a comparison of isolated white blood cells (WBCs) and Slingshot Biosciences’ FlowCytes® PBMC mimics, demonstrating true-to-life scattering and fluorescence.

Figure 1. Comparison of isolated white blood cells (WBCs) and Slingshot Biosciences’ cell mimics.

Applications of Slingshot Biosciences’ Cell Mimics

Slingshot Biosciences’ cell mimics support a variety of functions. “Our FlowCytes® Non-Fixed White Blood Cells serve as a forward and side scatter control for instrument standardization,” explains Barnette. “In addition, we offer TruCytes Biomarker Controls, which can be custom designed to express specific markers.” Other Slingshot products include ViaComp® Cell Health Controls, which bind to DNA intercalating dyes and amine-reactive dyes to simulate viability staining, and SpectraComp® Compensation Controls, which are single stain controls that optimize spectral unmixing results to reduce errors associated with spectral overlap.

Slingshot Biosciences in the Cell Therapy Process

Cell mimics have broad utility for cell manufacturing applications, as shown in Figure 2. For example, TruCytes Lymphocytes Subsets Cell Mimics can be used for defining cell subsets in patient samples, while custom TruCytes Biomarker Controls and SpectraComp Compensation Controls facilitate applications such as detecting rare biomarkers, monitoring cell isolation, and measuring transduction efficiency. “We’ve also recently launched TruCytes Potency Cell Mimics to support CAR T-cell development for CD19- or BCMA-based CARs,” says Barnette. “In In this setting, cell mimics with specific markers, namely CD19 or BCMA, are used as target cells in co-culture for functional potency assessments. In response to CAR T-cell binding, interferon-γ is generated, which can be measured by ELISA or a cytometric bead array assay. Slingshot’s technology enables the incorporation of additional CAR targets beyond BCMA and CD19 to create customized potency cell mimics.”

Figure 2. End-to-end applications of cell mimics across the cell therapy process.

Supporting Your Research

FluoroFinder has developed a suite of tools to help streamline your research. These include our Flow Cytometry Panel Design tool, which simplifies the process of selecting the best fluorophore combinations, and our Spectra Viewer, which lets you compare the spectral properties of more than 1,000 dyes alongside instrument-specific laser and filter configurations.

You might also want to refer to Slingshot’s extensive range of resources, which includes a case study reporting the use of custom TruCytes controls to validate a prognostic FcγRIIa flow cytometry test on fixed platelets and a recent Cytometry Part A publication that describes a reference control comparison.

*Slingshot Biosciences’ cell mimics are for research use only (RUO) and are not intended for use in clinical or diagnostic applications.

References:

1. Parums DV. A Review of CAR T Cells and Adoptive T-Cell Therapies in Lymphoid and Solid Organ Malignancies. Med Sci Monit. 2025;31:e948125. doi:10.12659/MSM.948125
2. Sharma A, Jaganathan BG. Stem Cell Therapy for Retinal Degeneration: The Evidence to Date. Biologics. 2021;15:299-306. doi:10.2147/BTT.S290331
3. Lu JS, Song CY, Cheng WJ, Wang KY. Mechanisms and challenges of mesenchymal stem cells in the treatment of knee osteoarthritis. World J Stem Cells. 2025;17(4):102923. doi:10.4252/wjsc.v17.i4.102923
4. Lin Q, Zhang S, Zhang J, et al. Colonic epithelial-derived FGF1 drives intestinal stem cell commitment toward goblet cells to suppress inflammatory bowel disease. Nat Commun. 2025;16(1):3264. doi:10.1038/s41467-025-58644-2
5. https://www.fda.gov/vaccines-blood-biologics/consumers-biologics/important-patient-and-consumer-information-about-regenerative-medicine-therapies
6. Zhao Y, Deng J, Rao S, et al. Tumor Infiltrating Lymphocyte (TIL) Therapy for Solid Tumor Treatment: Progressions and Challenges. Cancers (Basel). 2022;14(17):4160. doi:10.3390/cancers14174160
7. Parums DV. A Review of CAR T Cells and Adoptive T-Cell Therapies in Lymphoid and Solid Organ Malignancies. Med Sci Monit. 2025;31:e948125. doi:10.12659/MSM.948125
8. Salehi-Reyhani A, Ces O, Elani Y. Artificial cell mimics as simplified models for the study of cell biology. Exp Biol Med (Maywood). 2017;242(13):1309-1317. doi:10.1177/1535370217711441

High-content imaging (HCI) combines automated fluorescence microscopy with advanced image analysis to study cellular samples. This article looks at the key steps in a typical HCI workflow and explores some common applications. It also highlights some of the leading solutions available for HCI-based research, with insights from Andy Bashford, Imaging Product Marketing Manager at Molecular Devices, and Stephen Full, Senior Product Manager for Automated Microscopy Systems at Thermo Fisher Scientific.

What is High-Content Imaging?

When discussing high-content imaging, it is important to understand how it differs from three other interconnected practices: high-throughput screening (HTS), high-content screening (HCS), and high-content analysis (HCA). High-throughput screening is the process of testing high volumes of samples, such as large compound libraries or genetic mutations, for a specific biological response. Screening assays can be cellular or biochemical in nature (e.g., viability, marker expression, enzyme activity, binding interactions, reporter gene activity) and typically use miniaturization with automation to facilitate throughput.

High-content screening was developed to complement HTS, driven by the pharmaceutical industry’s need for a platform that could yield better, functional information in the discovery process¹. The first HCS platform, the ArrayScan®, was launched in 1997 by Cellomics, Inc. (now part of Thermo Fisher Scientific). This automated, cell-based imaging system enabled researchers to study compound effects on cell morphology, protein localization, and more by detecting multiple markers simultaneously with immunofluorescence^1,2. In recent years, advanced technologies including confocal optics, water immersion objectives, and AI-analysis tools have enhanced the capabilities of HCS systems to improve performance and enable a wider range of applications.

The terms high-content imaging and high-content analysis were coined to describe the capture and analysis of any cell image data, irrespective of whether it involved using an HCS platform. A defining feature of HCA is its use of sophisticated algorithms for quantifying cellular features from the acquired images, which can help link complex cellular phenotypes to in vivo behaviors.

The High-Content Imaging Workflow

The HCI workflow begins with preparing samples. Traditionally, these were fixed 2-dimensional (2D) cell cultures in microplates, but HCI is increasingly being applied to study live cells, 3D cultures, and tissue sections. To allow for visualizing specific cellular components or biomarkers of interest, the samples are incubated with fluorescent probes, such as nuclear stains or fluorescently-labeled antibodies. The microplates are then loaded into an automated microscope, often with the aid of a robotic plate handler to increase assay throughput. Following image acquisition, advanced HCA software is used to extract a wide range of quantitative measurements. These encompass cellular morphology, as well as protein expression levels and subcellular localization, and can reveal how cells respond to different treatments or stimuli.

High-Content Imaging Applications

Besides being used to augment high-throughput screening campaigns by providing functional information on cells, HCI supports many other applications. These include cellular pathway analysis, which can help determine the mechanisms underlying disease, and characterization of stem cells, which is important for regenerative medicine applications. Other HCI applications include assessing the efficiency and effects of gene delivery into cells, investigating neurite outgrowth, and studying the dynamics of viral infection. HCI is also used for toxicology studies based on measurements of cellular viability, apoptosis, or oxidative stress, which can help identify compounds that may pose a risk to human health.

Leading Solutions for High-Content Imaging

High-content imaging technologies vary in terms of the number of markers they can simultaneously detect, the level of throughput they can provide, and their ease of use. The following are some of the leading solutions:

ImageXpress® HCI.ai High-Content Screening System (Molecular Devices)

In January 2025, Molecular Devices launched their next generation imager, the ImageXpress HCS.ai High-Content Screening System. The platform is based on state-of-the-art AgileOptix technology, which combines proprietary, dual-spinning disk technology, an advanced solid-state light engine, and a sensitive scientific CMOS sensor to enable exceptional imaging of cells and subcellular components. “The all new MetaXpress Acquire software streamlines complex workflows, minimizing training times and helping the whole lab get the most out of the system,” reports Bashford. “AI-powered IN Carta® image analysis software leverages cutting edge tools to improve the accuracy and robustness of high-content image analysis.”

The ImageXpress HCS.ai offers multiple imaging modes: brightfield label-free imaging, widefield, and confocal fluorescent imaging. It also has a high-intensity laser light source option, enabling researchers to capture images faster with shorter exposure times and multiplex their experiments with 7 lasers and 8 imaging channels. Other noteworthy features of the ImageXpress HCS.ai include fully automated water immersion objective lenses and a magnification changer, which provides up to 12 magnifications in a single configuration. The system also offers a Deep Tissue Confocal Disk, designed to improve data quality from thick 3D samples, as well as Environmental Control for live cell experiments. “The flexible, modular design allows users to choose the right configuration and upgrade it in the future, letting them invest in a quality system that can grow as their needs develop,” says Bashford.”

Figure 1. ImageXPress HCS.ai High-Content Screening System.

ImageXpress® Pico Automated Cell Imaging System (Molecular Devices)

The ImageXpress Pico Automated Cell Imaging System offers researchers an accessible solution to high content imaging, combining high-resolution imaging with powerful analysis. The imager can run fluorescent and brightfield assays with 2.5X – 63X magnification, 6 filter positions, Z-stacking, Autofocus, Live Preview, and optional features such as Environmental Control and Digital Confocal 2D on-the-fly deconvolution, helping researchers to make the most out of their samples. “The system comes with over 25 pre-configured templates, ranging from simple cell counting to sophisticated neurite tracing,” reports Bashford. “The user-friendly, icon driven software makes it easier than ever to get started with high content imaging.”

CellInsight High-Content Screening Platforms (Thermo Fisher Scientific)

Thermo Fisher Scientific’s CellInsight High-Content Screening Platforms include the CellInsight CX5, which lets researchers analyze cellular samples in up to five fluorescent colors; the CellInsight CX7 LED Pro, which enables 7-color detection and is well-suited to time-lapse and live-cell imaging studies; and the CellInsight CX7 LZR Pro, which provides high throughput and supports 3D organoid and spheroid research involving thick specimens. “All of our CellInsight High-Content Screening Platforms offer high-speed image acquisition and powerful image processing algorithms, letting users parallelize image capture and analysis for multiplexed cytometry measurements in real time,” says Full. “To help researchers decide which platform best meets their needs, we’ve compiled key features, specifications, videos, and sample data on our website. We also suggest compatible reagents and assays for monitoring cell viability, proliferation, and function, or determining cell structure.”

Figure 2. Immunofluorescence image of a HeLa spheroid captured with the CellInsight CX7 LZR Pro. The sample was labeled with Ki67 antibody followed by Invitrogen Alexa Fluor 647 and Alexa Fluor 488 phalloidin and Hoechst 34580 stains for nuclei. Imaging was performed using a 10x objective and 70 micron pinhole setting.

EVOS Cell Imaging Systems (Thermo Fisher Scientific)

EVOS Cell Imaging Systems are designed to make high-end microscopy simple, supporting applications from basic cell culture through to advanced multiplex tissue imaging. “All of our EVOS microscopes can be self-installed and running within an hour,” comments Full. “Users have the option of using our basic instrument, the EVOS M3000 Imaging System, which is capable of two-color fluorescence and can automatically measure cell confluency in real time. Alternatively, for more demanding cell-based imaging applications, we offer the semi-automated EVOS M5000 Imaging System and the fully automated EVOS M7000 Imaging System. For researchers working within the spatial biology field, the EVOS S1000 Spatial Imaging System features spectral unmixing software that enables a single round of 9-plex multiplex immunofluorescence to be imaged faster than instruments utilizing cyclic technology.” To support applications involving high-content screening or analysis, Thermo Fisher Scientific has developed the EVOS M7000 HCA Package, which combines the M7000 instrument with Celleste 6 Image Analysis Software to simplify the interpretation of results.

Supporting Your Research

FluoroFinder has developed a suite of tools that can help streamline the design of your high-content imaging experiment. Use our Antibody Search function to find antibodies that are validated for immunofluorescence, then leverage our Spectra Viewer to confirm which dyes are compatible with your imaging system. For an in-depth look at the optical properties and spectral profiles of different dyes, check out our Fluorescent Dye Database for detailed information on more than a thousand different fluorochromes.

References: 

1. Taylor DL. A Personal Perspective on High-Content Screening (HCS): From the Beginning. Journal of Biomolecular Screening. 2010;15(7):720-725. doi:10.1177/1087057110374995
2. Way GP, Sailem H, Shave S, et al. Evolution and impact of high content imaging. SLAS Discov. 2023;28(7):292-305. doi:1016/j.slasd.2023.08.009

Recombinant antibody technology is addressing known limitations of traditional antibody molecules and opening up new areas of scientific research. We spoke with Michael Fiebig, Ph.D., Chief Scientific Officer at Absolute Antibody (a Vector Laboratories Company), Mary Mills, M.S., Product Manager, ZooMAb® Recombinant Antibodies at MilliporeSigma (the U.S. and Canada Life Science business of Merck KGaA, Darmstadt, Germany), and Michael Lyons, Ph.D., Product Manager, Recombinant Antibodies at Thermo Fisher Scientific to learn more about how recombinant antibodies are produced and the advantages they offer, as well as discuss state-of-the-art solutions for developing recombinant antibody products.

Methods for Producing Recombinant Antibodies

In general, there are three main routes for producing recombinant antibodies. First, an existing antibody sequence may be cloned into an expression vector, which is then introduced into host cells for in vitro antibody synthesis. Second, antibody display methods (e.g., phage, yeast, mammalian, or ribosome display) allow for identifying high-performing antibodies from a heterogeneous pool based on binding to an immobilized target. Third, single B cell screening technologies are used to isolate antigen-specific B cells from blood for screening and subsequent sequencing of optimal antibody clones.

“At Absolute Antibody, we sequence existing hybridomas, or use sequences derived via other methods, to produce antibodies in mammalian cells – typically HEK293, but occasionally CHO cells,”  explains Fiebig. “We deliberately opted against doing discovery ourselves as there is a wealth of established antibodies that have formed the backbone of decades of research. I believe that where antibodies already exist, we have an obligation to produce them recombinantly and assess their performance first, before deciding if new clones should be generated. This is to avoid creating a new angle to the reproducibility crisis where established methods using a specific clone cannot be accurately replicated due to a lack of availability of high quality material.”

MilliporeSigma produces its ZooMAb® recombinant antibodies using a proprietary B cell immortalization technology, which enables a high-yield recombinant expression system and facilitates the generation of a broad range of clones; these are then screened to identify the best-performing antibodies for further development. “Many of our ZooMAb recombinant antibodies are rabbit monoclonals since, compared to commonly used murine species, rabbits recognize a greater diversity of epitopes per antigen and produce a higher immune response to ‘difficult’ immunogens such as small molecules, peptides, and post-translationally modified proteins,” says Mills. “Importantly, ZooMAb recombinant monoclonal antibodies are developed with a sustainable eco-design approach, aimed to minimize the environmental impact.”

“ThermoFisher is pursuing multiple strategies to generate an expansive portfolio of recombinant antibodies,” reports Lyons. “These include converting existing hybridoma monoclonal antibodies into a recombinant format by sequencing high-performance clones, utilizing phage display libraries, and leveraging advanced technologies such as our Bigfoot Spectral Cell Sorter to perform large scale single B cell sorting on immunized animal models and identify favorable antibodies for recombinant expression. Additionally, we are using known antibody sequences to produce recombinant multiclonal antibody cocktails, called Superclonal Recombinant Antibodies. These provide the precision of recombinant antibodies with the benefit of multi-epitope recognition seen in polyclonal antibodies.”

Advantages of Recombinant Antibodies

Recombinant antibodies offer many advantages over traditional polyclonal and hybridoma-derived monoclonal antibodies. These include exceptional batch-to-batch reproducibility, which eliminates issues related to cell-line drift and mutations commonly observed in hybridoma production. Recombinant antibodies also exhibit enhanced specificity and sensitivity, enabling precise detection of target proteins in complex biological samples. Furthermore, because recombinant antibodies are generated from defined sequences, under tighter control compared to extracting antibodies from animal sources, their use can provide more reliable and reproducible results, as well as can mitigate against accidental hybridoma loss due to contamination or a freezer malfunction.

Another key benefit of recombinant antibodies is that they reduce animal use for research. Once the antibody sequence is obtained, production can be achieved using animal-free methods, with no need for animal-derived components (e.g., serum). Additionally, recombinant antibodies can enable faster production, although Fiebig cautions that while methods such as phage display take just a few weeks for selection, researchers should factor in extra time for antibody production and re-validation. Importantly, many further advantages can be gained as a result of recombinant antibodies’ vast capacity for engineering.

Recombinant Antibody Engineering

Recombinant antibody engineering has increased flexibility for established research applications, such as flow cytometry and IHC, as well as paved the way for developing novel techniques, including those based on the use of multi-specific antibodies. One of the best known approaches, species switching, involves changing the antibody backbone to that of a different animal. It can increase diversity in a panel of antibodies for multiplexing or be used to reduce immunogenicity for in vivo applications. Another popular strategy, class switching, alters an antibody’s isotype or subtype for reasons that include increasing avidity and improving in vivo effector function and stability.

Engineering can also be used to generate antibody fragments, such as Fab and scFv. “The smaller size of antibody fragments compared to their full-length counterparts lets them penetrate tissues more easily and clear more readily from animal systems through the renal pathway,” comments Mills. Antibody fragments also provide a means of abolishing Fc interference (the unwanted interaction between antibodies and Fc receptors expressed on the surface of immune cells), which may alternatively be addressed by introducing point mutations into the Fc region. “Our Fc Silent Antibodies contain key point mutations that abrogate Fc receptor binding, enabling researchers to reduce non-specific background in immunostaining methods and remove the antibody effector function in vivo,” says Fiebig.

Lyons adds that modifying the Fc region is a powerful tool Thermo uses to enhance antibody performance. Figure 1 shows testing data in immunocytochemistry for Thermo Fisher Scientific’s Parkin (A) and OCT4 (B) wild type and recombinant rabbit monoclonal antibodies, with the engineered products both demonstrating superior sensitivity over the wild type equivalents.

Figure 1. Testing data for Parkin (A) and OCT4 (B) recombinant rabbit monoclonal antibodies. Immunocytochemistry analysis of Parkin and OCT4 was performed using 70% confluent SH-SY5Y and NTERA-2 cells, respectively. The cells were probed with wild type (WT) and engineered Parkin or OCT4 Recombinant Rabbit Monoclonal Antibody at the indicated concentrations (Product # 740019R or 740020R). For Parkin, cells were also stained for nuclei, using SYTOX Deep Red Nucleic Acid Stain (Product # S11381).

Other engineering strategies include the addition of tags, which can aid purification, detection, immobilization, and  functionalization, and the process of humanization. “While humanization is commonly used to reduce immunogenicity, we have at times also applied our Prometheus humanization platform to overcome manufacturability and stability issues with research antibodies,” reports Fiebig. “This is not because the antibody needed to be human, but because many of the physical requirements of a humanized antibody in the clinic align with a good research tool antibody: reliable manufacture, low aggregation, high stability, and consistent performance.”

Future Perspectives

Demand for recombinant antibodies continues to rise, driven by factors including their superior quality, capacity for tailoring to specific targets or applications, and the reduced animal burden associated with their production. “Researchers are showing strong preference for our recombinant antibodies” says Lyons. “The benefits of recombinant antibody technology, combined with our advanced validation processes, provide a valuable assurance in the reliability and accuracy of our antibodies, which translates to more confidence in experimental results.”

Echoing these sentiments, Mills notes that the shift towards recombinant antibodies demonstrates a commitment to advancing research and therapeutic options in a more reliable and effective manner. “Recombinant antibody availability has significantly transformed the landscape of biomedical research and therapeutic development,” she says. “Today, recombinant antibodies are integral to drug development, diagnostics, and therapeutic interventions that are paving the way for more precise and effective treatments across a range of diseases.”

Supporting Your Research

If you’re looking for recombinant antibodies to advance your research, FluoroFinder can help! Our Antibody Search function enables you to quickly and easily find products that are validated for your chosen application. And, for fluorescence-based applications, our Spectra Viewer lets you to compare the spectral properties of more than 1,000 dyes alongside instrument-specific laser and filter configurations, simplifying the process of experimental design.