Best practices for antibody and fluorophore use safeguard assay performance

A defining feature of flow cytometry is its capacity to analyze single cells. This has led to its application across the entire drug development continuum, with recent advances in the field of immunotherapy driving the increased use of flow cytometry to support clinical trials. However, while best practices have been established for the validation of flow cytometry instrumentation, reagents, and methods, there are currently no standardized guidelines in place for performing flow cytometry in a regulated environment. To address the need for harmonization among different laboratories, various strategies have evolved to safeguard experimental reproducibility. Among these, critical reagent monitoring promises to reduce assay variability by providing tighter control over antibodies and fluorophores used for flow cytometry-based research. This article discusses the challenges of critical reagent monitoring for flow cytometry and suggests ways of ensuring reliable assay performance.

Challenges of critical reagent monitoring for flow cytometry

Broadly speaking, critical reagents are defined as those that contribute most to assay variability. In the case of flow cytometry, antibodies and fluorophores are considered to be critical reagents since their performance directly impacts the signal readout. Ensuring antibodies and fluorophores perform consistently over time presents inherent challenges, particularly as flow cytometry panels become larger and the number of commercially available fluorophores continues to grow. One factor that must be considered is the chemical stability of fluorophore-based reagents; tandem dyes especially have a limited shelf-life that restricts their long-term storage. The complexities of high-dimensional analysis also introduce complications. Because spectral overlap must be avoided, many labs choose to purchase multiple conjugates of the same antibody ready for later use; not only must each antibody conjugate be validated both individually and in the context of a multiplexed panel, but the expiration dates must be taken into account to avoid unnecessary waste. 


To determine how antibody and fluorophore reagents should be stored, researchers are advised to refer to the manufacturer’s datasheet. This should include the batch number and shelf-life of the product and should state whether aliquoting is recommended. Fluorophores should always be stored (and handled) away from direct light and, unless otherwise stated by the manufacturer, should never be frozen. Freezing should also be avoided for unlabeled antibody reagents. Where freezing is deemed necessary, freeze-thaw cycles should be kept to a minimum; the antibody manufacturer may also suggest adding a cryoprotectant such as glycerol. If reagents will be purchased in multiple batches over time, it is sensible to implement measures that ensure older products are used first; employing an electronic record system can help keep track of stocks.

Considerations for panel design

With modern flow cytometry being used to detect multiple targets simultaneously, it is critical that researchers take care with panel design. It is recommended that bright fluorophores be paired with scarce antigens, and vice versa, and that fluorophores be spread across as many lasers and detectors as possible to minimize the risk of spillover. The pH sensitivity of different fluorophores should also be considered, especially where fluorophores with different pH requirements will be combined in the same antibody diluent. Other fluorophore characteristics to bear in mind when constructing a multicolor panel include the excitation and emission maxima, and the Stokes shift; the latter can be exploited to allow fluorophores with similar excitation maxima to be combined in the same experiment. For longer duration flow cytometry studies, or studies requiring the use of strong lasers, it is important to select fluorophores that are resistant to photobleaching.

Minimizing lot-to-lot variability

Lot-to-lot variability can lead to inaccurate reporting and should always be assessed during critical reagent monitoring. For flow cytometry, such variability is typically due to inconsistent conjugation and/or staining intensities that can cause a shift in reagent performance. To minimize the risk of lot-to-lot variability, each new batch of reagent should be tested in-house (alongside an existing batch of reagent if appropriate) before being used with test samples. It is also suggested that, where possible, sufficient reagent of the same lot number is purchased to perform a complete study. Controls can rapidly alert end users to a potential change in reagent performance; flow cytometry controls include unstained, fluorescence-minus-one (FMO), and compensation controls, as well as known positive and negative samples.


All critical reagents should be accompanied by a certificate of analysis and/or a material data sheet to authenticate the source, quality, and any validated applications. It is recommended that end-users maintain an up-to-date electronic record detailing the purchase date, vendor, lot number, and expiration date of antibodies and fluorophores used for flow cytometry and that this includes any in-house validation data. Logging reagent use each time an experiment is performed can help identify potential sources of variation and can safeguard projects where reagents are shared by multiple operatives. Additionally, documenting the performance of refrigerated storage appliances over time helps ensure critical reagents are not compromised.

QC/QA considerations

A unique challenge for flow cytometry stems from the lack of quality controls with known concentration values, such as are used for ELISA. The user-dependent nature of data acquisition and analysis, and the number of different software systems available complicates matters further. When selecting QC material, it is important to consider how the data will be used; for assays supporting drug development and clinical trials, it is advised that both an internal control (e.g., a user-generated batch of cells enriched for the cell type of interest) and an external control bearing close resemblance to the test samples are included. Other quality considerations include maintaining accurate records of reagents, protocols, and instrument settings for complete traceability, and ensuring results align with pre-defined acceptance criteria.


Selecting critical reagents for flow cytometry

With critical reagents being pivotal to the success of any flow cytometry experiment, identifying the right products is key. Use FluoroFinder’s Spectra Viewer to quickly compare over 1000 fluorophores from all suppliers in one intuitive platform, or turn to our Panel Builder to optimize your multiplexed experiment with the very latest fluorophore and antibody offerings across >60 suppliers. Whether you’re engaged in early drug discovery, or you’re involved in running a clinical trial, FluoroFinder simplifies experimental design for results you can rely on.