Why can your cytometry results be wrong without you knowing it?

Flow cytometry is an extremely powerful tool for cell analysis, but it is also surprisingly easy to make mistakes that compromise your results, even without realizing it. A bad setup, an inadequate gating strategy or lack of proper controls can generate inaccurate or even completely wrong data.

If you have ever wondered why your results are not reproducible or do not match what you expected, this article is for you.

1.Errors in Gating: The beginning of all evils

Gating defines how you select cell populations in your data. Incorrect or inconsistent gating can cause you to misinterpret the proportion or activation of certain cells.

Common problems:

• Not excluding debris or doublets.
• Using hand-drawn gates without objective criteria.
• Changing the order of gates in different experiments.

Tip: Use standardized strategies and always check negative controls to define the correct placement of your gates.

2. Lack of controls: The invisible enemy

One of the most frequent errors in cytometry is not including appropriate controls, which prevents the data from being interpreted correctly.

Essential controls:

• Non-specific fluorescence (NSF): To define true positivity limits.
• Isotype controls: To validate antibodies.
• Unlabeled cells: To establish background autofluorescence.

3. Detector saturation: More is not always better

A common mistake is to use antibodies in excess, thinking that this will improve the signal. What actually happens is detector saturation, which completely distorts intensities and marker ratios.

Consequences:

• Loss of resolution between populations.
• False positives due to spillover.
• Non-linear fluorescence curves.

Solution: Titrate your antibodies correctly to determine the optimal concentration. Using more reagent than necessary not only worsens the result, but increases the cost of the experiment.

4. Misapplied compensation: The chaos of the multicolor panel

In multicolor panels, incorrect compensation can lead to serious artifacts. Signals get “mixed” between channels and end up generating falsely positive or negative data.

Recommendations:

• Use compensation beads for each fluorochrome.
• Compensate before applying gating.
• Verify compensation values, especially if you change the laser or gain.

Conclusion: Cytometry needs more than just pressing “RUN”

Errors in cytometry can go unnoticed if you don’t know where to look. Poorly raised gating, missing controls or system saturation can ruin even the best designed experiment.

At Immunostep, we offer not only validated reagents for flow cytometry, but also expert technical advice to help you optimize your workflow and avoid these common mistakes.