Electrophoretic cytometry of adherent cells

Abstract

Cell-matrix and cell-cell interactions influence intracellular signalling and play an important role in physiologic and pathologic processes. Detachment of cells from the surrounding microenvironment alters intracellular signalling. Here, we demonstrate and characterise an integrated microfluidic device to culture single and clustered cells in tuneable microenvironments and then directly analyse the lysate of each cell in situ, thereby eliminating the need to detach cells prior to analysis. First, we utilise microcontact printing to pattern cells in confined geometries. We then utilise a microscale isoelectric focusing (IEF) module to separate, detect, and analyse lamin A/C from substrate-adhered cells seeded and cultured at varying (500, 2000, and 9000 cells per cm2) densities. We report separation performance (minimum resolvable pI difference of 0.11) that is on par with capillary IEF and independent of cell density. Moreover, we map lamin A/C and β-tubulin protein expression to morphometric information (cell area, circumference, eccentricity, form factor, and cell area factor) of single cells and observe poor correlation with each of these parameters. By eliminating the need for cell detachment from substrates, we enhance detection of cell receptor proteins (CD44 and β-integrin) and dynamic phosphorylation events (pMLCS19) that are rendered undetectable or disrupted by enzymatic treatments. Finally, we optimise protein solubilisation and separation performance by tuning lysis and electrofocusing (EF) durations. We observe enhanced separation performance (decreased peak width) with longer EF durations by 25.1% and improved protein solubilisation with longer lysis durations. Overall, the combination of morphometric analyses of substrate-adhered cells, with minimised handling, will yield important insights into our understanding of adhesion-mediated signalling processes.

Laterally Aggregated Polyacrylamide Gels for Immunoprobed Isoelectric Focusing

“…Comparison to other published IEF performance benchmarks suggests that the 2% PEG highly porous PA gels offer ΔpI min performance on par with or exceeding that of commonly used formats, including capillary IEF 45 and others (benchmark gels, ΔpI min = 0.13 ± 0.02 using Polybuffer ampholytes over a pH 4−7 gradient; 31 IEF in free solution, ΔpI min = 0.11 for adherent-cell IEF platforms using Zoom ampholytes over a pH 4−7 gradient 46 ). Concurrent IEF analyses of pI markers and tGFP isoforms allow estimates of IEF peak capacity, n c , which reports the number of wellseparated protein bands in a single IEF separation lane (n c = L/4σ, where L = separation lane length, 4σ = peak width).…”

“…The n c for IEF was 12.0 ± 5.9 in the benchmark gels, 16.9 ± 7.9 in the negative control gels, and 19.0 ± 10.2 in the 2% PEG highly porous gels (across 3 replicates each, Supplemental Figure S5). Given the success of the benchmark gels for immunoprobed IEF of complex biospecimens, , the n c suggests that the 2% PEG highly porous gels are suitable for high-performance IEF, which is important for resolving protein isoforms and post-translational modifications.…”

In Situ Single‐Cell Western Blot on Adherent Cell Culture

“…To evaluate the tunability of the one‐step fabrication approach for diverse cell culture needs, we fabricated in situ scWB devices using a range of applied FN* concentrations (1–100 μg mL −1 , determined based on previous work) and microwell diameters (50–100 μm). Using confocal imaging, we observe FN* localized to the surface of all devices ( n =4).…”

Multi-parametric cell profiling with a CMOS quad-modality cellular interfacing array for label-free fully automated drug screening

“…Multi-modality label-free time-lapse cell transparency and cell-to-surface adhesion measurements of on-chip cultured fibroblasts Cell-to-surface adhesion and cell transparency are critical phenotypic parameters for functional drug screenings and drug toxicity/safety assessments. 11,23,42,[46][47][48][49][50][51] Cell-to-cell and cell-to-extracellular-matrix (ECM) adhesive properties are closely related with important physiological processes, such as tissue organization, cell viability and proliferation, 49 and cell migration. 50 Moreover, cell adhesion assays are essential to investigate the activation of membrane-bound proteins and interactions with extracellular microenvironments, 51 which play central roles in disease pathogenesis, such as cardiac fibrosis and cancer metastasis, wound healing, and therapeutic target identification.…”