Troubleshooting Tip 6: I want to assess cell viability, but do not know where to start.

 

Problem

This image shows one of the standard, most widely used ways to assess cell viability. Cells (insect cells in the example shown) are mixed with Trypan Blue, a dye which stains dead cells, and then mounted in a specialized chamber of a known volume known as a hemacytometer. Living and dead cells are then counted on a brightfield microscope, and cell viability (perecentage of living cells) is determined.

Possible Causes and Solutions

Methods for assessing cell viability are essentially based on probes that report on:

1. the permeability of the plasma membrane or/and

2. the physiological state of the cell.

Assays for the permeability of plasma membrane

To probe cell membrane integrity, one mixes cells with a vital dye. Healthy cell membranes are impermeable to large or charged molecules and will thus exclude the dye. The dye will however penetrate dead/unhealthy cells with damaged membranes and stain the cells

One of the most widely used dyes is Trypan Blue. Dead/unhealthy cells that take up Trypan Blue are easily identified as dark blue on a brightfield microscope, are directly counted and the viability of the culture determined. Alternatively, one can use fluorescent dyes such as propidium iodide (PI) or 7-AAD which label DNA of membrane-damaged cells. Cells that take up PI or 7-AAD are hundreds or thousands of times brighter than healthy cells, and are typically counted by flow cytometry.

Assays for the physiological state of the cell

To assay physiological properties of the cells, one typically uses lipophilic reagents that enter the cells and report for a metabolic process or ion gradients at mitochondrial membranes.

A standard reagent that reports on a metabolic process is carboxyfluorescein diacetate (CFDA). CFDA is nonfluorescent and diffuses into cells. However, once in the cell FDA gets cleaved nonspecifically by intracellular esterases to generate a fluorescent product that is charged and is thus retained in the cell. Living cells are then typically counted by flow cytometry. Alternatively, one can use reagents such as Rhodamine 123 that are sensitive to membrane potential. Rhodamine 123 is a lipophilic dye that partitions to mitochondria membranes thus staining healthy mitochondria bright green.

Details on how to assess cell viability can be found in Current Protocols in Cytometry, Unit 9.2, Current Protocols in Neuroscience, Unit 7.18 and Current Protocols in Immunology, Appendix 3B. Information on basic mammalian cell culture techniques including cell counting and assessment of cell viability can be found in Current Protocols Essential Laboratory Techniques, Unit 4.3 and Current Protocols in Cell Biology, Unit 1.1.

Visit www.currentprotocols.com for tools, calculators, apps, videos, and information on all Current Protocols methods.

Contributed by: Manos Mavrakis.