FluoroSpot assays are indispensable tools for immunologists seeking to quantify cytokine secretion at the single-cell level. With their multiplexing capabilities, high sensitivity, and compatibility with a wide range of immune and secreting cell types, FluoroSpot technology offers insights into polyfunctional immune responses and quantification of single-cell secretion of cytokines. However, achieving reproducible and biologically meaningful results requires careful attention to assay setup and execution.

In this post, we highlight five common issues in FluoroSpot workflows and provide practical troubleshooting tips to help researchers optimize their experiments.

1. No Spots Present

• Cell Viability: Ensure PBMCs or other cell types are >90% viable. Resting cryopreserved cells post-thaw can restore functionality.
• Positive Controls: Use validated recombinant protein-based controls. A diffuse fluorescence signal (not discrete spots) confirms proper assay setup.
• Cell Number: ~10,000 PBMCs per well is a good starting point. Titrate based on expected cytokine frequency, stimulation conditions, and cell type.
• Incubation Time: Cytokine secretion kinetics vary. Adjust incubation based on your target analyte.
• Stimulation Concentration: Optimize stimulation dose to avoid underwhelming responses.

2. Too Many Spots Present

• Cell Density: Reduce cell numbers to prevent overlapping spots and saturation.
• Stimulation Intensity: Excessive stimulation may cause non-specific activation. Titrate to refine specificity.

3. Fuzzy or Poorly Defined Spots

• Incubator Stability: Avoid moving plates during incubation. Even minor shifts can blur spot morphology.
• Plate Stacking: Incubate plates in a single layer to ensure uniform temperature distribution.

Impact of well disturbance during cell plating. The well from a disturbed plate shows uneven cell distribution and irregular spot morphology, which can compromise signal clarity and quantification.

4. Spots Localized to the Sides of the Well

• Pipetting Technique: Avoid adding reagents post-plating. This can push cells to the periphery.
• Cell Addition Timing: Pre-mix cells with stimulation reagents before plating. Or, add your cells to the well as the final step.
• Centrifugation: Briefly centrifuge plates to evenly settle cells, this is especially important during short incubation times.

Comparison of FluoroSpot wells with and without centrifugation. The well from a plate subjected to centrifugation shows a more uniform cell distribution and clearer spot formation, while the well from a non-centrifuged plate exhibits uneven cell settling and diffuse signal, potentially impacting assay interpretation.

5. Artifacts in Wells

• Blotting Materials: Use lint-free paper towels to avoid fiber contamination when blotting plates after washing.
• Automated Washing: Manual washing introduces variability. Automated washers improve consistency.
• Buffer Quality: Use a 0.22 μm sterile filter for critical buffers (e.g., Coating Buffer A, Assay Diluent B, Wash Buffer) to remove particulates and prevent microbial growth.

Summary

FluoroSpot assays offer unmatched resolution in cytokine profiling and immune cell function analysis, but small missteps can lead to misleading results. By refining your technique and optimizing key parameters, you can ensure your data is both reproducible and biologically meaningful.

FluoroSpot MAX™ Deluxe is compatible with imaging cytometers and other fluorescence imaging platforms capable of whole-well fluorescence imaging, making this assay accessible to more labs without traditional FluoroSpot readers. 

For examples on instrumentation troubleshooting, explore our comprehensive user guide for FluoroSpot MAX Deluxe analysis using the Revvity Celigo™ Image Cytometer.

Our FluoroSpot MAX Deluxe Sets provide researchers with pre-optimized reagents, streamlined workflows, and high-sensitivity detection, empowering your immunology research and discovery.