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Updated: Dec 21, 2025
IP-FCM: Immunoprecipitation Detected by Flow Cytometry
Published on: December 2, 2010
FACSCanto II and LSRFortessa flow cytometer instruments can be synchronized utilizing single-fluorochrome-conjugated
Annelisa M Cornel1, Christine A J van der Burght1, Stefan Nierkens1
1Center for Translational Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.
Insights
Synchronizing flow cytometers FACSCanto II and LSRFortessa is crucial for consistent results. Single-fluorochrome beads improve synchronization between these instruments, ensuring reliable immunophenotyping data for clinical diagnostics.
Area of Science:
- Immunology
- Biotechnology
- Clinical Diagnostics
Background:
- Multiparameter flow cytometry is essential for cell immunophenotyping across diverse sample types.
- Standardization of flow cytometry is critical to minimize inter-laboratory variability and subjective interpretation.
- Instrument synchronization is indispensable for achieving comparable results in clinical diagnostic settings.
Purpose of the Study:
- To evaluate the effectiveness of calibration bead-based synchronization between FACSCanto II and LSRFortessa flow cytometers.
- To determine optimal bead types for achieving instrument comparability.
Main Methods:
- Two FACSCanto II and two LSRFortessa instruments were synchronized using multicolor hard-dyed and single-fluorochrome-conjugated surface-dyed beads.
- Whole-blood samples from healthy controls were stained and analyzed post-synchronization.
Main Results:
- Comparability criteria (<15% deviation) were met for same-model instrument synchronization with both bead types.
- FACSCanto II and LSRFortessa synchronization failed with multicolor hard-dyed beads but succeeded with single-fluorochrome beads.
- Single-fluorochrome beads allowed for successful synchronization and stricter comparability criteria.
Conclusions:
- Single-fluorochrome-conjugated surface-dyed beads effectively synchronize FACSCanto II and LSRFortessa instruments when multicolor beads fall short.
- This synchronization method reduces inter-instrument deviations, enabling more stringent comparability standards in flow cytometry analysis.
Background:
Multiparameter flow cytometry is the preferred method to determine immunophenotypic features of cells present in a wide variety of sample types. Standardization is key to avoid inconsistencies and subjectivity of interpretations between clinical diagnostic laboratories. Among these standardization requirements, synchronization between different flow cytometer instruments is indispensable to obtain comparable results. This study aimed to investigate whether two widely used flow cytometers, the FACSCanto II and LSRFortessa, can be effectively synchronized utilizing calibration bead-based synchronization.
Method:
Two FACSCanto II and two LSRFortessa flow cytometers were synchronized with both multicolor hard-dyed and single-fluorochrome-conjugated surface-dyed beads according to the manufacturer's instructions. Cell staining was performed on five whole-blood samples obtained from healthy controls and were analyzed upon synchronization with the respective synchronization protocols.
Results:
Comparability criteria (defined as <15% deviation from the reference instrument) were met with both bead sets when synchronizing different FACSCanto II or LSRFortessa instruments. However, we observed that the criteria could not be met when synchronizing FACSCanto II with LSRFortessa instruments with multicolor hard-dyed beads. By utilizing single-fluorochrome-conjugated surface-dyed beads to determine and adjust PMT voltages, the accepted comparability criteria were successfully met. The protocol has been validated using five different eight-parameter stained samples.
Conclusion:
We show that FACSCanto II and LSRFortessa instruments can effectively be synchronized using single-fluorochrome-conjugated surface-dyed beads in case deviation criteria cannot be met using multicolor hard-dyed beads. Synchronization with single-fluorochrome-conjugated surface-dyed beads results in decreased deviations between instruments, allowing comparability criteria to become stricter.
