Guava Express Software Module From Guava Technologies

Guava Express is one of several modules contained within the Guava Cytosoft program suite that comes with Guava’s personal cell analysis systems. My experiences are with the version of software bundled with the Guava PCA 96 System. This particular module is designed for measuring intracellular or cell surface marker expression; however, it can be adopted for a range of assays as it bears close resemblance to software common to other flow cytometers. Hence, it is one of the more flexible modules offered with the PCA 96 System.

I used the system to screen for cell surface marker expression associated with osteogenesis (principally STRO-1 and alkaline phosphatase). I screened both human mesenchymal stem cells and MG-63 human osteosarcoma cells for these markers, predominantly in dual-staining experiments after culturing the cells in conditions to induce differentiation. I used unconjugated primary antibodies which were then probed with secondary antibodies usually conjugated to either R.Phycoerythrin or Phycoerythrin-Cy5. During the acquisition of the samples on the PCA 96 system, I used the Guava Express module to appropriately gate the cells and afterwards, analyze the relative proportion of cells expressing my proteins of interest.

The major pros and cons of this software module align with the benefits and limitations of the instrument itself. Its strongest feature is the simplicity with which protein expression can be quantified. Essentially, the program uses a single-screen “dashboard” of the flow cytometer functions, simplified for ease of use. Following assay-dependent cell staining procedures and preparation of sample for flow cytometry, the user follows simple sequential set-up screens designed to appropriately adjust the cytometer settings for data acquisition. Used in conjunction with the in-depth application guide, users with minimal flow cytometry experience will find the system simple to use for protein quantification or to compliment other assays previously conducted (such as Western blotting, ELISAs or immunofluoresence). Performing operations such as gating your cell population and changing the marker position are all readily available on the acquisition screen control without the use of pull-down menus. Dot plots and histograms are also automatically loaded for ease of use. Information such as flow rate, cell count and statistics of results based on marker position are also automatically displayed on the screen. The system simplifies the acquisition of data by stripping away all complicated operations not normally required by most users, leaving only the bare minimum control and information required to achieve accurate quantification of fluorophore emission from cells. In short, advanced knowledge of flow cytometer software is not required.

However, the major element not present on this particular system which compromises assay accuracy is the ability to compensate for signal cross-over when both fluorescence channels are used. The effect of signal cross-over on the accuracy of the assay depends to some extent on the fluorophores used and the lasers chosen for your system. Given these issues, I feel it is generally best that users limit the use of labels to single color experiments. The use of a single color also further simplifies data acquisition.

It should also be mentioned that Guava’s technical support is extremely helpful in recommending fixation and staining procedures to help ensure your assay is optimally designed. This is particularly useful if you are new to flow cytometry and do not have access to experienced personnel in your organization.

If you are looking for more advanced features, newer versions of Guava Express (Guava ExpressPro and ExpressPlus) may overcome some of these limitations, as well as offering additional features. However, I have not used these versions of the software and cannot verify this. To maximize the data obtained from your assays and allow for the greatest flexibility in your assay design, you will probably find more benefit trying a more conventional flow cytometer. While it may be more difficult initially, a conventional flow cytometer may be more suitable to your assay requirements.