A new tool for routine testing of cellular protein expression: integration of cell staining and analysis of protein expression on a microfluidic chip-based system

Abstract

The key benefits of Lab-on-a-Chip technology are substantial time savings via an automation of lab processes, and a reduction in sample and reagent volumes required to perform analysis. In this article we present a new implementation of cell assays on disposable microfluidic chips. The applications are based on the controlled movement of cells by pressure-driven flow in microfluidic channels and two-color fluorescence detection of single cells. This new technology allows for simple flow cytometric studies of cells in a microfluidic chip-based system. In addition, we developed staining procedures that work "on-chip," thus eliminating time-consuming washing steps. Cells and staining-reagents are loaded directly onto the microfluidic chip and analysis can start after a short incubation time. These procedures require only a fraction of the staining reagents generally needed for flow cytometry and only 30,000 cells per sample, demonstrating the advantages of microfluidic technology. The specific advantage of an on-chip staining reaction is the amount of time, cells, and reagents saved, which is of great importance when working with limited numbers of cells, e.g., primary cells or when needing to perform routine tests of cell cultures as a quality control step. Applications of this technology are antibody staining of proteins and determination of cell transfection efficiency by GFP expression. Results obtained with microfluidic chips, using standard cell lines and primary cells, show good correlation with data obtained using a conventional flow cytometer.

FIGURE 1

Chip layout and features. The microfluidic glass chip is fixed in a plastic caddy which accommodates six sample wells (green), two buffer wells (grey), one well for a reference dye (light green), and one well for a vacuum interface and collection of fluid waste (orange). A common buffer channel joins each sample channel in close proximity to the detection area (marked in red).

FIGURE 2

On-chip antibody staining results are displayed as dot plots for all six samples. Each dot represents one cell displayed at two fluorescent values on logarithmic scale. Red region marks double positive cell population. The 293 cells and a CD86 expressing cell clone were harvested and washed and cell density was adjusted to 3 million cells per milliliter in an isobuoyant cell buffer. Positive and negative control (sample 1 and 6) and four mixtures of parental and transfected cells were prepared at different ratios. Ten microliters of cell suspension were incubated with calcein-AM and anti hCD86-APC antibody (prediluted in 1:8 in CB) directly in the chip wells.

FIGURE 3

Comparison between on-chip CD86 antibody staining results from four different experiments. The data were obtained with classically stained samples measured on a conventional flow cytometer. Data are in good agreement with those obtained by the reference technique and the theoretical prediction.

FIGURE 4

Optimization of transfection conditions for GFP. CHO-K1 cells were transfected with GFP DNA at various DNA:lipofectamine ratios (1:2 to 1:10) or with lipofectamine in the absence of DNA (control). Cells were then stained on-chip with the live-cell stain CBNF and analyzed on the 2100 bioanalyzer (A). Blue fluorescence histograms of CBNF positive cells and percentages of transfection are shown. The same cells were stained with CBNF according to conventional procedures and analyzed on a flow cytometer (B) (10,000 events were measured per sample).

FIGURE 4

Optimization of transfection conditions for GFP. CHO-K1 cells were transfected with GFP DNA at various DNA:lipofectamine ratios (1:2 to 1:10) or with lipofectamine in the absence of DNA (control). Cells were then stained on-chip with the live-cell stain CBNF and analyzed on the 2100 bioanalyzer (A). Blue fluorescence histograms of CBNF positive cells and percentages of transfection are shown. The same cells were stained with CBNF according to conventional procedures and analyzed on a flow cytometer (B) (10,000 events were measured per sample).

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