Flow Cytometry Issues and Solutions

Common problems that occur during flow cytometry sample preparation.

By Cheryl Lacy

Published on October 31, 2024

Flow cytometry is a powerful technique used across research and clinical laboratories to analyze cell populations, protein expression, and other cellular features. However, the quality and reproducibility of flow cytometry data heavily relies on precise sample preparation. Even small errors during this foundational step can lead to high background, data inaccuracies, and compromised data quality. Yet, upstream sample preparation is often overlooked as researchers focus on optimizing later steps in the other workflow like antibody staining. It is not only important that researchers ensure that the sample preparation techniques they are using suit the cell type and marker they are evaluating, but also that they consider how their washing method(s) affect their cells.

Below are some common flow cytometry challenges that improved sample preparation practices can help avoid or mitigate.

High Background and Non-Specific Binding

High background and non-specific antibody binding can obscure the population of interest and result in ambiguous or skewed data.

Solution: Increasing the wash buffer volume or adjusting the number and/or duration of washes can help reduce background. Switching to gentler washing methods can minimize dead cells, a common cause of higher background and non-specific antibody binding.

Cell Loss During Washing

Traditional cell washing methods, such as centrifugation and manual methods, can cause cells to be lost or damaged. This leads to variability and inaccurate cell counts, especially with fragile or low abundance cells.

Solution: Adjusting centrifugation speeds may help prevent cell damage, and manual washing is often used as an alternative when working with fragile cell types. However, some researchers recommend no-wash protocols or techniques like magnetic cell separation to protect sensitive or rare cell populations. These methods account for cell loss or damage during washing steps by requiring cell density levels of approximately 0.5 million cells per mL during analysis to offset the expected volume loss.

But what if you no longer needed to compensate with additional cells or could better protect your fragile cells during the washing step?

Cell Viability Issues

Dead or dying cells can introduce data artifacts and skew population statistics. These compromised cells are more likely to uptake nonspecific stains, further complicating analysis.

Solution: Shortening sample processing times and using gentler methods can help to reduce cellular stress, while viability dyes help to distinguish live cells from dead. Handling your samples on ice or at 4°C also supports cell health.

High Fluorescent Intensity

When excess amounts of antibodies are trapped in the sample, abnormally high fluorescence intensity can occur.

Solution: Adequate washing with the addition of detergents like Tween^® 20 or Triton^® X-100 reagents to the wash buffer can reduce trapped antibodies. Automated methods help maintain consistent reagent mixes, minimizing the risk of errors [too high or low a mix or forgetting to add the mix].

Other Common Issues

High Signal In Negative Cell Populations: Additional wash steps between antibody incubations help to reduce signal artifacts.
Incomplete Red Blood Cell Lysis: This may require extra washes [up to three between each step] to remove red blood cell debris.

Each of these issues has a common factor: optimizing the washing step in sample preparation can drastically reduce these challenges. High quality sample preparation is the foundation of any successful study, ensuring reproducible quantitation and reliable data.

Cell Washing Techniques

Centrifugation, the most common cell-washing method, introduces mechanical stress that can lead to cell damage, cell loss, and increased debris. Researchers often resort to manual washing for a gentler approach though it brings its own challenges, including user-to-user variability, lots of time, and user ergonomics concerns, and it’s difficult to ensure that debris removal is as complete as possible.

The introduction of Curiox Laminar Wash technology changed the landscape of sample preparation by providing a gentler, consistent wash process. Building on this, the release of the Curiox C-FREE™ Technology in Summer 2024 offers an advanced approach to sample washing and liquid handling.

C-FREE Technology: Automating Gentle Cell Washing

C-FREE technology supports automation and workflow optimization by using a laminar flow process to gently washes cells, and removes debris and unbound antibodies. It reduces mechanical stress and cell loss associated with centrifugation. By minimizing manual steps, C-FREE technology also reduces variability and enhances consistency across experiments.

In Summary

Effective sample preparation is the foundation of successful flow cytometry analysis. By addressing common issues in sample preparation, researchers can minimize artifacts, improve data reproducibility, and gain more accurate insights from their experiments. With C-FREE technology, you can more confidently enhance your sample preparation and achieve better data quality and streamlined workflows. Consider the possibilities:

What if you no longer needed to allocate extra cells to ensure the density required for your downstream applications?
What if your wash mix was automatically and consistently managed?
And what if variability from researcher to researcher was eliminated?

Learn more about how C-FREE technology can address these challenges. Click here to request a sales representative contact you for a consultation. Discover more about Curiox technology.