Multiparameter analysis of apoptosis using lab-on-a-chip flow cytometry.

Area of Science:

• Biotechnology
• Cell Biology
• Analytical Chemistry

Background:

• Microfluidic flow cytometry (µFCM) integrates miniaturized systems for cell analysis.
• Conventional flow cytometry offers multiparameter data but requires larger sample volumes.
• Advancements in chip-based cytometers enable high-content analysis with reduced sample input.

Purpose of the Study:

• To present innovative protocols for analyzing cell death and cell cycle using a microfluidic flow cytometry system (Fishman-R).
• To demonstrate the utility of specific fluorescent probes for tracking cellular events in real-time.
• To enable multivariate analysis of cellular processes in small sample volumes.

Main Methods:

• Development of protocols for microfluidic flow cytometry (µFCM).
• Utilized DRAQ7 as a plasma membrane-permeability marker.
• Employed PhiPhiLux for caspase activation detection.
• Used TMRM and DRAQ5 probes to assess mitochondrial membrane potential and DNA content, respectively.

Main Results:

• Successfully tracked caspase activation during programmed cell death using PhiPhiLux and DRAQ7.
• Monitored dissipation of inner mitochondrial membrane potential with TMRM in conjunction with DRAQ7.
• Correlated mitochondrial membrane potential changes with cell cycle profiles (DNA content) in leukemic cells using TMRM and DRAQ5.
• Measured caspase activation alongside cell cycle profiles in tumor cells using PhiPhiLux and DRAQ5.

Conclusions:

• The Fishman-R microfluidic flow cytometry system effectively analyzes complex cellular processes.
• Integrated protocols allow for simultaneous assessment of cell death markers and cell cycle status.
• µFCM offers a powerful, miniaturized platform for multiparameter cell analysis with low sample volumes.