Evaluating extracellular matrix influence on adherent cell signaling by cold trypsin phosphorylation-specific flow...

Area of Science:

• Cell Biology
• Biochemistry
• Immunology

Background:

• Tissue microenvironments, rich in extracellular matrix (ECM) proteins, modulate cellular responses to growth factors.
• Studying microenvironmental effects on cell signaling often involves in vitro culture of adherent cells on ECM-coated surfaces.
• Phosphorylation-specific flow cytometry quantifies single-cell signaling but requires trypsinization, which can activate cell signaling pathways.

Purpose of the Study:

• To develop a method for analyzing intracellular phosphorylation-dependent signaling in adherent cells without trypsin-induced artifacts.
• To enable accurate measurement of cell signaling responses influenced by the extracellular matrix in single adherent cells.

Main Methods:

• Developed a cold trypsin-phosphorylation-specific flow cytometry protocol.
• Cells were prepared for analysis on ice (~0°C) to maintain trypsin activity while inhibiting intracellular kinases.
• Quantified intracellular phosphorylated extracellular signal-regulated kinase (pERK) levels in primary human vascular smooth muscle cells.

Main Results:

• The cold trypsin method preserves intracellular kinase inactivity while allowing trypsin to facilitate cell preparation.
• Successfully quantified intracellular pERK levels in single adherent primary human vascular smooth muscle cells cultured on different ECMs.
• Demonstrated the method's utility for analyzing cell signaling in response to microenvironmental cues.

Conclusions:

• The cold trypsin-phosphorylation-specific flow cytometry method is generally applicable to various adherent cell types, including primary patient-derived cells.
• This technique leverages the temperature-dependent activity of trypsin to enable robust analysis of cell signaling.
• Provides a valuable tool for measuring microenvironmental influences on single-cell signaling in adherent cells.