Introducing the Dish Soap Protocol: A Unified Approach for Multi-Modal Intracellular Staining

Area of Science:

• Cellular Biology
• Immunology
• Biochemistry

Background:

• Multidimensional flow cytometry allows for extensive cellular analysis.
• Current fixation protocols present challenges in simultaneously preserving intracellular targets and nuclear antigens.
• Balancing membrane permeabilization and structural integrity is crucial for intracellular staining.

Purpose of the Study:

• To evaluate the impact of various fixation and permeabilization reagents on key cellular features.
• To develop a cost-effective buffer solution for enhanced multidimensional flow cytometry.
• To enable simultaneous detection of intracellular proteins and nuclear targets without compromising cell integrity or fluorescence.

Main Methods:

• Assessment of fix-perm reagents on nuclear staining, fluorescent protein retention (e.g., GFP), cytokine detection, epitope preservation, and cell viability.
• Development and testing of a novel buffer formulation, "Burton's Best Buffer".
• Comparison of the novel buffer with commercial fixation buffers.

Main Results:

• Common fixation protocols demonstrate trade-offs between nuclear accessibility and intracellular target preservation.
• The novel "Burton's Best Buffer" effectively preserves fluorescent proteins and allows for nuclear staining.
• The developed buffer maintains cell integrity, scatter profiles, and fluorophore stability while enabling detection of transcription factors and cytokines.
• "Burton's Best Buffer" is significantly more cost-effective than commercial alternatives.

Conclusions:

• A novel, cost-effective fixation buffer overcomes limitations in multidimensional flow cytometry.
• Simultaneous detection of intracellular and nuclear targets is achievable, enhancing cellular analysis.
• This protocol supports advanced flow cytometry applications, including cytokine and transcription factor analysis.