Overcoming fixation and permeabilization challenges in flow cytometry by optical barcoding and multi-pass...

Area of Science:

• Cell Biology
• Immunology
• Biotechnology

Background:

• Cell fixation and permeabilization are crucial for intracellular biomarker detection in flow cytometry.
• These chemical processes can compromise the integrity of fragile targets like cell surface proteins and fluorescent labels, impacting data accuracy.
• Current flow cytometry methods face limitations due to the destructive nature of sample processing.

Purpose of the Study:

• To present a novel multi-pass flow cytometry technique for analyzing delicate cellular components.
• To overcome the limitations of conventional flow cytometry by enabling sequential measurement of the same cells.
• To improve assay flexibility and data quality in cell analysis workflows.

Main Methods:

• Utilizing individual cell barcoding with laser particles for sequential cell analysis.
• Measuring chemically-fragile markers and fluorochromes before fixation and permeabilization.
• Integrating measurements of intracellular markers post-fixation with pre-fixation data.

Main Results:

• Accurate measurement of intracellular fluorescent proteins and methanol-sensitive antigens/fluorophores.
• Successful analysis of both surface and intracellular markers without compromising data integrity.
• Demonstration of single-cell resolution maintained throughout the multi-pass analysis.

Conclusions:

• The multi-pass flow cytometry approach enhances assay flexibility and enables comprehensive cell analysis.
• This technique overcomes the constraints of traditional one-time measurement flow cytometry.
• The innovation offers significant potential for applications in immuno-oncology, stem cell research, and cell biology.