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Published on: August 6, 2025
Iterative restaining as a pivotal tool for n-color immunophenotyping by slide-based cytometry
Wiebke Laffers1, Anja Mittag, Dominik Lenz
1Department of Otorhinolaryngology/Surgery, University of Bonn, D-53105 Bonn, Germany.
Summary
Slide-based cytometry enables detailed immunophenotyping of peripheral blood leukocytes (PBLs) by allowing cells to be analyzed multiple times. This iterative restaining method enhances multi-color analysis capabilities.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Traditional flow cytometry is limited by the number of parameters that can be analyzed simultaneously.
- Slide-based cytometry (SBC) offers a unique approach to re-interrogate cells after initial analysis.
Purpose of the Study:
- To explore the utility of SBC for detailed immunophenotyping of peripheral blood leukocytes (PBLs).
- To assess the feasibility of iterative restaining for multi-color analysis in SBC.
Main Methods:
- Peripheral blood leukocytes (PBLs) were stained for CD-markers and DNA.
- Cells were immobilized on a glass slide and analyzed using laser scanning cytometry.
- Iterative restaining was performed for subsequent analyses, with data merged at the single-cell level.
Main Results:
- Six virtual immunostainings were achieved by repurposing the PE-channel for four CD-markers.
- This demonstrated the capacity for extensive marker analysis on the same cell population.
Conclusions:
- Iterative restaining is a powerful technique within SBC for advanced n-color immunophenotyping.
- SBC concepts, including iterative restaining, may become pivotal for complex cellular analysis.
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