Analysis needs more than excitation and detection

A simple, multiplex assay for simultaneous analysis of transfection efficiency, cell count and cell viability

Transfection of DNA into foreign hosts is a common laboratory procedure used to analyse the function of gene products in the context of living cells. Most transfection procedures involve formation of DNA-lipid complexes, followed by fusion with cells and introduction of DNA into the nucleus. While fairly routine, this process still requires optimisation of assay conditions for different cell types, and monitoring of the efficiency of DNA introduction.
There are a variety of methods now available to monitor transfection efficiency in cell populations, most of which follow the expression of a fluorescent, luminescent or colourimetric gene product. The method presented here uses one such reporter gene, Green Fluorescent Protein (GFP), which provides the added benefit of reporting transfection in living cells without the need for fixation, or the addition of indicator dyes or reagents. Furthermore, it is possible to multiplex additional indicators with GFP to report on apoptosis, cytotoxicity, or even a second auto-fluorescent protein.
Analysis of GFP expression requires instrumentation capable of fluorescence excitation and emission detection, and is commonly performed using methods such as manual microscopy analysis, fluorescence plate reader detection, or traditional flow cytometry. Each method has its associated features and benefits for development and optimisation of transfection assays, but the Guava platform provides a powerful alternative that uses a small volume of cells and provides comparable data quality to more complex, time-consuming, and expensive approaches. For example, unlike plate readers, the Guava system provides information on individual cells and, compared to microscopy, the Guava system does not require manual counting methods, which become complicated and tedious when dealing with multiple fluorophores. Overall, the Guava platform provides an ideal solution for evaluating multiple transfection conditions in a microplate format, and delivers the added flexibility to monitor aspects of the transfection beyond the efficiency of gene transfer, such as cell viability or induction of apoptosis.

Results
The Guava platform provides a simple and robust system for single-cell analysis that integrates a microcapillary sampling system with intuitive software that displays and interprets multi-parameter data. In the data shown in Figure 1, the user is able to easily distinguish live, dead and transfected cells based upon a colour-coded data display, and the numerical data is summarised both onscreen and in a spreadsheet format for offline analysis. The data shown were generated using the Guava PCA-96 AFP system, which provides both a blue (488nm) laser excitation optimised for GFP analysis, and the added benefit of microplate compatibility for increased assay throughput and walk-away automation. Furthermore, the quality of the data generated by the Guava platform for transfection efficiency is claimed to be equivalent to that generated using traditional flow cytometry equipment.

The system design is ideal for assay development to determine the optimal combination of cell concentration, DNA amount, and volume of transfection agent that will yield the best transfection efficiency, expression level, and lowest possible toxicity. The data shown in Figure 3 represents a broad matrix of transfection conditions in an attempt to identify the ideal combination for Jurkat cells, which are traditionally difficult to transfect.

Figure 3. Multiplex transfection analysis using a 96-well microplate.

Using a 96-well plate and GFP as the indicator, it was possible to set-up a series of assays on one microplate and analyse the cells over multiple days. Furthermore, the Guava system provides the range of multi-parameter assay outputs to provide the quality information needed to make better decisions (Table 2). In this specific example, cell health was determined by monitoring the cell concentration on subsequent days rather than using propidium iodide, but the assay could just as easily be run with a specific indicator of viability included, taking full advantage of the platform flexibility.

Table 2. Guava Assay Output(s)
Assay Output                        Utility
Cell Concentration                                                                          Percent Positive, GFP         Measurement of transfection efficiency
Mean Intensity, GFP           Positive Cells Indicator of expression level
Percent Positive, PI            Measurement of cytotoxicity
Percent Positive, PI                           

The Guava platform provides high accuracy and sensitivity for determination of transfection efficiency, with the added benefits of microplate compatibility, minimal training and maintenance, and the ability to multiplex assays for assessment of important features such as cell viability or continued cell proliferation. In addition, users are able to increase productivity in the lab by saving time and allowing the instrument to operate unattended, while also reducing costs through reduced reagent and cell usage.

Data kindly provided by Dr. Linda Jacobsen, Roche Applied Science, Indianapolis, IN, USA
enquiry number 05101