Flow cytometry has become more accessible to the life science community in recent years thanks to the development of smaller, more sophisticated instruments coupled with their relative affordability. One such company to capitalize on this growing trend is Guava, now part of Millipore, and their EasyCyte flow cytometry systems. The advantage these systems currently enjoy is their ease-of-use through the intuitive software modules and their pre-packaged reagent kits that require minimal optimization. The standardization of fundamental assays (e.g. cell viability, proliferation, and apoptosis) now offers novice users the ability to run and analyze sophisticated flow cytometry assays with minimal experience and training.
The ViaCount Reagent is used to determine both cell count and cell viability. The principle of the assay is the differential permeability of two DNA-binding dyes; the nuclear dye binds DNA in all cells, whilst the second viability dye exclusively binds the DNA in dead cells. Apoptotic cells are distinguished from viable and dead cells based on the respective stronger and weaker binding of the viability dye. Cellular debris is excluded from the count since cell fragments lack DNA and so do not readily bind either dye.
The reagent is supplied as a ready-made solution that is added directly to suspensions of single cells followed by a brief incubation period. Since we routinely work with multiple cell samples and conditions, we use a 96-well plate, which allows us to utilize a multi-channel pipette thus significantly increasing workflow compared to multiple single tubes. Despite the light sensitivity of the reagent we did not observe significant differences in counting if the reagent was used on the bench in ambient light or in a darkened environment. One of the most important steps to obtain accurate, reproducible counts is ensuring the even suspension of the cells during dilution, staining and immediately prior to loading on to the Guava instrument. We achieve an even suspension through gentle up and down pipetting; vortexing is not recommended as it may disrupt the cells and reduce the viability of the cell population being analyzed. Since the assay requires a certain number of cells (1 x 104 to 5 x 105) to obtain reliable results, it is recommended that a dilution series is performed the first time this assay is run on a new cell type.
We have successfully used the ViaCount Reagent for analyzing spleen and thymus single cell suspensions using a Guava EasyCyte flow cytometer. The integration of the software module and the reagent make it extremely easy to run the samples and analyze the data. The gates can be adjusted so you can analyze the viable, apoptotic, and dead cell populations, which are reported in percentage, number, and graphical format. Overall, the ViaCount reagent allows the accurate determination of cell counts and viability of cell samples with minimal manipulation and hands-on time.
Project Scientist
Molecular, Cellular and Developmental Biology
University of California, Santa Barbara