Are You Choosing the Right Cell Viability Assay?

Viability is a ubiquitous endpoint in cell-based assays. The results make a general claim about the current state of the cells well-being which can then be used to calculate important metrics like a test compound’s LD50 or the functional state of an organ which will be transplanted. Viability assays can be divided into three groups; membrane permeability, mitochondrial assays, and functional tests.

  • Membrane permeability assays rely on the fact that dead cells will have a permeant cell membrane. Dyes like trypan blue or propidium iodide will cross compromised membranes marking the cells as dead. Related assays take advantage of the changes a cell undergoes as the membrane breaks down. The LDH assay detects an enzyme that is released into media. Fixable Live/Dead cell impermeant dyes take advantage of the greater number of available amines to increase fluorescent signal as cell membranes break down.
  • Mitochondrial assays use the redox potential of viable cells to reduce viability indicators into measurable metrics. Resazurin is broken down into resorufin in the colorimetric and fluorescent MTT assay. Realtime-Glo similarly uses the reduction potential of cells to break a pro-substrate down into a substrate which can bind with a luciferase enzyme creating light.
  • Functional assays are broader and include cell motility, contractility, or morphology; the ability of cells to adhere, divide, or maintain an ion gradient; and total ATP or hemoglobin content.

Although each assay is attempting to get at a similar answer, it is important to be aware that the different methods can lead to different answers. To show this in action, Visikol OpenLiver spheroids were grown for one week before being treated with a gradient of acetaminophen for 48 hours. After which cell viability was measured with Fixable Live/Dead Green (Thermo) an assay of membrane permeability, Realtime-Glo (Promega) an assay of mitochondrial potential, and Celltiter-Glo (Promega) an assay of cell function.


Visikol OpenLiver spheroids were treated with a gradient of acetaminophen for 48 hours. The acetaminophen was added via half-exchange. Each concentration and condition were repeated in triplicate. The Fixable Live/Dead dye was added by half exchange for 30 minutes before fixation. The Celltiter-Glo was added by equilibrating the spheroids to room temperature before adding the reagent and measuring luminescence 20 minutes later. The Realtime-Glo was added by half exchange 2 hours before measuring the luminescence.

The percentage of cell viability for the Celltiter-Glo and Realtime-Glo are reported as percentages of the vehicle. The percentage of cell viability for the Fixable Live/Dead was calculated by measuring the percentage of each spheroid belly slice which contained positive fluorescent staining and then comparing each calculated percentage to the vehicle. This was done to remain consistent with the method of calculating viability used for the luminescent indicators. The vehicle control surface area percentage was calculated as 78%.


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