The Challenge of Distinguishing Cell-Cell Complexes from Singlet Cells in Non-Imaging Flow Cytometry and Single-Cell...

Area of Science:

• Immunology
• Cell Biology
• Biotechnology

Background:

• Flow cytometry and cell sorting are crucial for single-cell analysis.
• Interpreting single-cell data requires careful consideration of potential artifacts.
• Cellular doublets or complexes can be erroneously included in singlet gates.

Purpose of the Study:

• To identify strategies for distinguishing cell-cell complexes from true singlets in flow cytometry data.
• To address the misinterpretation of mixed-lineage gene signatures caused by cellular contamination.
• To provide methods for accurate analysis of single immune cells.

Main Methods:

• Analysis of T cell-monocyte and T cell-B cell complexes using flow cytometry.
• Utilizing molecular signatures at protein and mRNA levels for differentiation.
• Employing imaging flow cytometry and post-sorting microscopy for detection.
• Applying developed strategies to a published dataset.

Main Results:

• T cell-monocyte and T cell-B cell complexes exhibit distinct molecular signatures.
• Imaging flow cytometry and microscopy effectively detect cell-cell complexes.
• A previously reported novel cell population was identified as likely T cell-B cell complexes.
• Strategies successfully distinguished complexes from singlets.

Conclusions:

• Careful gating and validation are essential when interpreting flow cytometry data.
• Cell-cell complexes can be reliably identified using molecular and imaging techniques.
• This work prevents misinterpretation of single-cell data and the discovery of artifactual cell types.