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 (pMLC^S19) 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.

Fig. 1

In situ IEF measures protein peaks from single and clustered cells adhered to a substrate gel. (A) Concept schematic of in situ IEF (not-to-scale). Step 0: Cells are cultured on pre-determined regions by seeding cells on the ECM-patterned substrate gel. Step 1: A chemically-imprinted lid gel, which contains the lysis reagents and carrier ampholytes, is interfaced with the substrate gel, which contains the cells attached to the ECM-patterned region, for 30 s. Step 2: An electric potential (600 V) is then applied for 6 min, establishing a stable, linear pH gradient. Step 3: After focusing, proteins are covalently immobilised in the substrate gel via UV activation of a benzophenone moiety. Step 4: Gels are then washed and immunoprobed for targets of interest. Individual protein peaks are then quantified. (B) Inverted fluorescence kymograph of pI markers incorporated into the substrate gel and focused. The established pH gradient is linear and stable for >20 min. (C) Left: False-colour inverted fluorescence micrograph of micropatterned rhodamine-fibronectin, onto which cells were cultured, lysed, separated via IEF, photocaptured, and immunoprobed for lamin A/C. Right: Fluorescence intensity profile of lamin A/C protein peaks that have passed the SNR > 3 and Gaussian fit R² > 0.7 threshold. Black line indicates Gaussian fit. IEF, isoelectric focusing. pI, isoelectric point. ECM, extracellular matrix. AFU, arbitrary fluorescence units.

Fig. 2

The substrate gel contains a tuneable, ECM-patterned region onto which cells adhere. (A) False-colour inverted micrographs of gels patterned with varying geometries and pattern sizes of rhodamine-fibronectin. The SNR of the smallest circular feature (d = 30 μm) was 55. (B) Brightfield images of cells seeded at varying densities. The density of cells controls the cell-to-cell spacing. (C) Cells cultured at a starting concentration of 500 cells per cm² on the fibronectin-patterned gels proliferate over a period of four days, quadrupling in cell number. Error bars represent the standard deviation. Scalebars, 500 μm. SNR, signal-to-noise ratio.

Fig. 3

In situ IEF separates and detects proteins from single and clustered cells. (A) Brightfield images of single cells and a ~80% confluent pattern of cells prior to in situ IEF (left), and false-colour inverted fluorescence micrographs of lamin A/C that is retained on the fibronectin pattern and focused into a Gaussian peak (middle). The protein peaks are segmented and fluorescence intensity profiles are generated for each peak (right). Black line indicates Gaussian fit. (B) Separation performance, as measured by lamin A/C peak width, does not significantly change with higher seeding density, indicating that protein is not overloaded. (C) Protein mass measurements of lamin A/C that is retained on the fibronectin pattern (ID) and focused (AUC). Bars represent the mean. Scalebars, 200 μm. AFU, arbitrary fluorescence units. ID, integrated density. AUC, area under curve. pI, isoelectric point.

Fig. 4

Protein expression does not correlate with cell morphology. (A) Brightfield micrographs of cells of varying area and corresponding fluorescence intensity profiles of lamin A/C and β-tubulin. (B) Comparison of lamin A/C and β-tubulin protein content (AUC and ID), which correlate poorly with cell area, as measured via brightfield. (C) ICC fluorescence micrograph of fluorescently-labelled (Cytopainter) cells seeded on patterned gels. Lamin A/C protein expression does not correlate with cell area. (D) Scatter plots of lamin A/C protein mass as a function of cell circumference, form factor, eccentricity, and cell area factor (cell area/form factor). Lamin A/C protein mass did not correlate with these morphometric parameters. Scalebars, 100 μm. ICC, immunocytochemistry. AFU, arbitrary fluorescence units. ID, integrated density. AUC, area under curve.

Fig. 5

In situ IEF of adherent cells assays protein profiles that differ from trypsin-treated (detached) cells. (A) False-colour inverted fluorescence micrographs and intensity profiles of IEF of single cells detached using trypsin and seated into microwells (left) or analysed via in situ IEF of fibronectin-patterned (attached) cells (right). (B) Box plot of GFP control protein content (AUC) of focused peaks indicates comparable expression between microwells and fibronectin-patterned cells. Line in the box represents the median and box ends represent the 25th and 75th percentiles. Red dots indicate outliers. (C) Box plot of β-integrin indicates higher expression in patterned cells. (D) Comparison of measured SNR of various targets. Compared to microwell IEF, in situ (adhered cells on fibronectin pattern) IEF results in higher SNR of β-integrin, pMLC^S19, and CD44, targets that are adversely affected by trypsin treatment. The red line indicates SNR threshold of 3. Scalebars, 200 μm. AUC, area under curve. SNR, signal-to-noise ratio. AFU, arbitrary fluorescence units. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Fig. 6

Separation performance, as measured by lamin A/C peak width, and protein solubilisation are enhanced by tuning the EF and lysis durations, respectively. (A) False-colour inverted fluorescence micrographs and intensity profiles of single cells lysed for 30 s and electrofocused for 6 min, compared to either a longer EF duration of 8 min, or longer lysis durations of 45 s or 70 s. Contrast adjusted for improved visualisation. Black line indicates Gaussian fit. (B) Analysis of peak width for longer EF duration indicates improved separation performance (lower peak width). Bars represent the mean. (C) Comparison of lamin A/C protein mass retained on the fibronectin pattern or focused to lamin A/C pI as a function of EF duration. (D) Comparison of lamin A/C protein mass retained on the fibronectin pattern or focused to lamin A/C pI as a function of lysis duration. The longest lysis duration (70 s) improved lamin A/C solubilisation, as evidenced by higher focused protein content. Data from 30 s lysis duration are the same as the 6 min EF duration. Scalebars, 200 μm. AFU, arbitrary fluorescence units. ID, integrated density. AUC, area under curve. EF, electrofocusing.