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Measuring Cell Viability

Besides cell counting, measuring cell viability is probably the most common procedure, in the cell biology laboratory. A cell viability assay or test should determine whether the cells are alive or dead. If too many dead cells are present, the cell suspension should be "cleaned up" using a method that removes the dead cells.

There are two types of viability assay. These are:

  • Dye exclusion viability assays
  • Metabolic viability assays

Dye Exclusion Viability Assays

As its name implies, a dye exclusion assay uses a dye or stain that can enter the cell and usually intercalates with the DNA in the nucleus. The mere entry of the dye into the cell assumes that the cell membrane has lost its integrity and that the cell is dead. In other words, live cells should exclude the dye, while dead cells allow the dye to enter. Dyes or stains that are used in dye exclusion viability assays include, but are not limited to:

  • Typan blue, often used to detect both viability and cell number using a hemocytometer or similar in an automated instrument.
  • Propidium iodide (PI), which can be detected using manual and automated techniques, including a flow cytometer. This stain is also used for cell cycle analysis and other assays.
  • 7-Aminoactinomycin D (7-AAD), usually detected by flow cytometry and often used in cellular therapeutic applications to measure the CD34+ cell content.
  • Acridine Orange, often used in hemocytometer procedures, but can also be detected by flow cytometry. This is a toxic compound.

Metabolic Viability Assays

Metabolic viability assays do not rely on the assumption that the cell membrane must loose its integrity in order to determine whether a cell is alive or dead. Metabolic viability assays usually rely on the cell's ability to perform a specific biochemical reaction that can be measured usually by absorbance, fluorescence or luminescence methods. These might include:

  • Reduction of a tetrazolium compound, e.g. MTT/XTT and measured by absorbance.
  • Conversion of non-fluorescent Calcein AM to a fluorescence-emitter after acetoxymethyl ester hyrolysis by intracellular esterases.
  • Function of ion pumps and channels detected by fluorescence.
  • Change in pH indicators detected by fluorescence.
  • Cellular and mitochondrial metabolism detected by measuring ATP using luminescence methodology.


Metabolic Viability Assays from HemoGenix®

All HemoGenix® assays that measure intracellular ATP production or MTS tetrazolim reduction in the mitochronia of cells are metabolic viability assays. They include:

  • LIVEGlo™
  • All HALO®, ImmunoGlo™, MSCGlo™ and STEMGlo™ assays
  • All HemoLIGHT™, ImmunoLight™, MSCLight™ and STEMLight™ assays.


Thus, with the exception of LIVEGlo™, all other assays simultaneously measure metabolic viability and cell proliferation or cytotoxicity.



Discrepancies Between Dye Exclusion and Metabolic Viability Assays


Click image for larger view

Discrepancies Between Dye Exclusion and Metabolic Viability Assays

Dye exclusion viability assays do not detect cellular functionality. This is the primary difference between a dye exclusion and metabolic viability assay. It is this difference that can lead to discrepancies between the viability methodologies. It is also this difference that can lead to a false interpretation of results.

Consider the following example. Many cellular therapeutic laboratories measure viability by staining cells with 7-AAD and flow cytometry. This method is used because, in hematopoietic stem cell processing laboratories, viability can be easily combined with other cell membrane markers, such as CD34 and CD45 in a single flow cytometric enumeration panel. This type of viability testing is regularly used for bone marrow, mobilized peripheral blood and umbilical cord blood stem cell products. Virtually all cord blood products are cryopreserved and can remain in the frozen state for many years. In the example shown in the accompanying graph, 56 cryopreserved cord blood samples were thawed, the mononuclear cells (MNC) fractionated by density gradient centrifugation and tested for viability using 7-AAD as well as measuring the proliferation, cellular functionality and viability using HALO®-96 SPC-QC. The discrepancy between dye exclusion and metabolic viability methodologies is readily apparent. In this example, HALO™-96 SPC-QC is used to simultaneously detect the viability and cell proliferation of primitive hematopoietic stem cells (SC-GEMM, equivalent to CFC-GEMM in the CFU assay).

The results indicate that although the 7 out of the 54 total number of samples were below the 85% cut off level (FDA Guidance for Industry. Minimally Manipulated, Unrelated Allogeneic Placental/Umbilical Cord Blood Intended for Hematopoietic Reconstitution for Specified Indications) for assuming high viability and therefore cell growth, 56% or about 30 of the stem cell samples were metabolically dead and therefore could not demonstrate stem cell proliferation. In addition, all of the remaining 44% of the samples showed intracellular ATP values less than 0.25µM ATP, which is between 50-75% less than if the cord blood samples were fresh.


This discrepancy between dye exclusion and metabolic viability was again noticed for another cohort of cord blood samples studied and published in the Journal of Translational Medicine in 2015.