From an aspiring biologist’s first laboratory course in college, the hemacytometer with its embedded grid pattern becomes a basic tool of his or her trade. Biologists spend countless hours over the course of their training and careers staring at these grids through a microscope. Were this not enough of a validation of one’s dedication to their field, the trypan-blue dye exclusion chemistry routinely used for cell counting requires mastering the scientific art of interpreting and classifying particles based on their size and “blueness” characteristics.

The interpretation of these multiple shades of blue or blue-gray results in subjectivity and imprecision, particularly when technician-to-technician factors are considered. In addition, trypan blue chemistry has been reported in the literature to consistently
overestimate cell viability.1, 2

Over the last few years Guava Technologies say they have developed a significant following around the world with their automated Guava ViaCount assay for cell counting and viability assessment. The ViaCount assay is run on the Guava PCA instrument, an automated platform for cell-based assays. The chemistry is based on a combination of two nucleic acid dyes used to identify cells while excluding cellular debris, and to distinguish live cells from dead. This approach also offers the unique ability to simultaneously assess the apoptotic cell fraction within a sample.

Also of note is the fact that the assay protocol calls for only 20μl of the original cell sample, conserving often-precious cells. This contrasts with the 1ml of sample consumed by other automated cell counting systems, 50 times the volume used by the Guava system.

The system consists of the Guava PCA instrument, a laptop computer with the ViaCount software module, and the ViaCount reagent. The system provides a total cell count, viable cell count and percentage, and optionally, an apoptotic cell count and percentage. The system does not require an additional reagent to assess the apoptotic cells.
The PCA instrument is based on capillary flow cell technology, enabling rapid analysis of individual cells. The detection system is laser-based for high sensitivity.

The assay bases its results on a 3-parameter correlated assessment of each “event” detected by the system. The size of each particle passing through the flow cell is measured along with its fluorescence in two spectrally distinct channels or wavelengths. The ViaCount reagent includes two DNA intercalating dyes. The first of these dyes is membrane permeant, entering and staining all nucleated cells. The second dye is a viability dye which penetrates and stains dead and dying cells with compromised membrane integrity, and is excluded by cells with intact membranes.

Guava has shown that cells which take up intermediate amounts of the viability dye also react with Annexin V, indicating they have entered the apoptotic pathway. In comparison with trypan blue-based methods, the Guava ViaCount assay provides a more accurate and complete evaluation of cell cultures claim the company.

Guava also offers other assays available for the platform. Each of the Guava

For more detailed assessment of apoptosis across a broad range of applications, Guava offers apoptosis assays for Annexin V, Caspases, and

TUNEL. This growing range of assays is designed to make the most critical cellular assays accessible to life science researchers everywhere.

Most of these additional assays have traditionally only been possible using on a flow cytometer, an expensive and complex technology usually requiring a specially trained, dedicated operator working in a central flow cytometry facility. Consequently accessing this wide range of valuable cellular analysis data has necessitated the considerable inconvenience of scheduling central flow cytometry facility time well in advance of your experiment.

In a large number of laboratories worldwide the Guava ViaCount assay has been proven to offer an improved and more comprehensive alternative to traditional methods for cell counting and viability assessments. In addition, say the company, the availability of a wide and growing menu of optimised cell-based assays for the platform, offers scientist and technicians a simple, compact and affordable system which provides access to capabilities previously out of reach for most laboratories.