Electrophoretic cytometry of adherent cells.

Cell adhesion influences intracellular signaling, but detachment for analysis disrupts these signals. Traditional methods require cell detachment, which alters protein expression and phosphorylation states. Prior research has shown that enzymatic treatments can disrupt receptor proteins and dynamic phosphorylation events. This gap motivated the development of a method that preserves cell-substrate interactions during analysis. No prior work had resolved how to analyze adherent cells without detaching them. Existing techniques lack the ability to correlate morphometric data with protein expression in situ. The need for a non-disruptive analysis method persists in proteomic studies. This paper introduces a novel approach to address these limitations.

The aim of this study is to develop and characterize a microfluidic system for analyzing adherent cells without detaching them. The specific problem is the disruption of cell signaling during detachment. The motivation is to preserve signaling integrity and receptor protein expression. The authors propose using microcontact printing and isoelectric focusing (IEF) for this purpose. The system allows for in situ analysis of cell lysates. The study tests whether this approach can maintain cell morphology and signaling. The goal is to correlate morphometric features with protein expression. This method could improve understanding of adhesion-dependent signaling.

The researchers used microcontact printing to pattern cells in defined geometries. They then applied microscale isoelectric focusing (IEF) to analyze lysates from adherent cells. Cell densities of 500, 2000, and 9000 cells per cm² were tested. Lamin A/C and β-tubulin proteins were separated and detected. Morphometric parameters like cell area and form factor were measured. The IEF module was optimized for separation performance. Lysis and electrofocusing durations were varied to assess solubilization. The system was validated for its ability to preserve cell attachment during analysis.

The IEF module achieved a minimum resolvable pI difference of 0.11. This performance was consistent across all tested cell densities. Lamin A/C and β-tubulin expression showed weak correlations with morphometric data. CD44 and β-integrin proteins were better preserved in adherent cells. Phosphorylated pMLC^S19 was detectable only in adherent cells. Longer electrofocusing durations improved separation by 25.1%. Extended lysis durations enhanced protein solubilization. These results suggest the method preserves signaling integrity better than traditional approaches.

The study concludes that the microfluidic system enables in situ analysis of adherent cells. This approach preserves cell-substrate interactions and signaling states. The IEF module performs reliably at various cell densities. Morphometric data alone poorly predict protein expression. The method enhances detection of receptor proteins and phosphorylation events. Longer electrofocusing improves separation performance. Longer lysis durations improve protein solubilization. These findings support the use of this system for adhesion-dependent signaling studies.