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Posted on Thu, Jun 20, 2019

Cell based assays

Cell based assays are used to quantify cellular function, measure how stimuli affect cells, or to localize an effect within the cell. The cells are live and intact, and require the use of fluorescent tags and chemiluminescent or colorimetric enzymes. The quantification is performed by flow cytometry or microscopy. This is very different from studies of protein or nucleic acid which require destruction of the cell and isolation of those components from cell lysate. A cell based assay is conducted entirely within live, intact cells. The goal is to understand a cellular process, localization of a molecule or drug to a cellular compartment, or to measure how cells react to a substance. Cell based assays are usually performed in tightly controlled cell lines to test for a wide range of behaviors:

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  • cell viability: ratio of live to dead cells—used to understand drug effects or to find the best culture conditions for the cells
    • redox potential
    • ability of cell membrane to stay intact
    • enzymatic activity
    • trypan blue or calcein-AM
  • cell proliferation: growth rate of the cell population
    • number of cellular divisions
      • N-succinimidyl ester is cleaved into a fluorescent product upon cell division and the intensity decreases by half with each division
    • DNA synthesis
  • cytotoxicity: how many cells die after treatment with a drug candidate?
  • cell senescence: specific tags for markers of cell senescence
  • cell death
    • apoptosis- caspase 3 activity is commonly measured
    • autophagy
    • necrosis
  • cell organelle labeling: nucleus, nuclear envelope, nucleoli, microtubules/cytoskeleton, centrosomes, endoplasmic reticulum, plasma membrane, peroxisomes, mitochondria, golgi, intracellular junctions

 

Cell based assays most often use immortalized cell lines instead of primary cell lines because primary cell lines can behave very differently from batch to batch. However, immortalized lines can lose some of the character of “real” cells. Another problem with primary cells is that they cannot be passaged more than a few times without displaying phenotypic changes or entering senescence. The repeatability of the assay is most important, so cell established lines are carefully selected for the type of assay being conducted. Commercial cell based assay kits usually only contain the fluorescent tags and chemiluminescent or colorimetric enzymes needed for the assay along with an experimental protocol and tips for assay interpretation. The cells used may be selected for and cultured  by the customer, which is a labor-intensive process, or can be purchased. There are thousands of established and characterized cell lines commercially available. These lines have been passaged from an extensive sampling of tissues from a variety of species.

Cell based assays are particularly important to the drug development process. It is important to test a drug candidate in well-defined cell culture conditions before moving on to live animals or later stage clinical trials. Many drugs fail in late stage clinical trials because of unanticipated side effects in more complicated systems. Cell based assays can’t prevent this, but they can help to identify problematic drugs early in development.


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