We are continuing our blog with a review of cell death assays for drug discovery. In this posting I will describe cellular metabolism assays to use in your project to measure cell death, cytotoxicity, and proliferation. All assays in our blog are available as a service from Alera Labs. Please contact us at (919) 228-8209 to learn more about our assays services.

Cell Death Assays Measuring ATP or Mitochondrial Metabolism

Quite simply, these assays are based on the assumption that living cells have active mitochondrial metabolism and produce ATP.

Luciferase Assays to measure ATP

Using ATP as a surrogate marker for viability was made possible in high-throughput screening format by luciferase-based assays popularized by Promega Corporation (1):

Figure 1. Firefly Luciferase produces light by promoting reaction between Luciferin and ATP in the presence of oxygen.

Luciferase-ATP reagents are quite costly (at ~$1/assay as of 2011) while also requiring access to expensive (and exotic for many labs) luminometer or scintillation counter. But Luciferase-ATP assays excel where high sensitivity is required. It is possible to assay ATP levels in less than 100 cells and in our hands we saw measurable signal from less than 50 cells. The signal is proportional to the number of cells present. If your cells are truly precious or small numbered, Luciferase assays are a way to go. For example we used this assay for stem cell research projects as well as cell migration project where only a small number of cells migrated across semi-permeable layer.

Luciferase assay hands-on:

The assay is simple to use and requires only a single addition of reagent directly to cells in culture medium. The procedure does not require additional handling steps, such as removal of medium or washing of cells, making luciferase easy and convenient to use.

Promega is the company that markets most of the luciferase reagents. It is probably possible to prepare homebrew version of luciferase assay, but for most projects it is more feasible to buy ready-to-use reagents from Promega.
CellTiter-Glo Luminescent Cell Viability Assay is ATP measurement kit for cell death research. To perform the assay, prepare CellTiter-Glo Reagent by reconstituting the lyophilized CellTiter-Glo Substrate in CellTiter-Glo Buffer. Add the Reagent in a volume equivalent to the amount of medium in which the cells are plated. Mix for 2 minutes, and after a 10-minute incubation, detect the emitted luminescence using a plate-reading luminometer. In the past we had good experience with BMG Labtech dedicated luminometers, but multimode microplate readers with luminescence capability would work as well unless you need high sensitivity of detection. Alternatively, a CCD camera may be used to record the luminescence.

Tetrazolium Salt Conversion

For truly universal assays to measure cytotoxic, antiproliferative, and cell death effects don’t look further than tetrazolium salt conversion assays. Most common assays available include MTT, XTT, or WST-1 (e.g. from Roche Applied Science LINK). They are colorless salts that are readily taken by the cells and converted  onto colored products by mitochondrial reductases. Again, this method assumes that mitochondrial respiration is a good surrogate marker for cell viability. Luckily this is true for most but not all cases, with limitations discussed below.

Figure 2. Principle of MTT assay. Colorless tetrazolium salt on the left is being converted by respiring mitochondria into colored formazan product on the right.

These assays are very useful for measuring of non-lethal perturbations to cell well-being including senescence, starvation, contact inhibition and less than ideal growth conditions because cellular metabolism is roughly proportional to cell proliferation. Number of cells present can be estimated as well because 2 cells in a similar state can convert tetrazolium salts at twice the rate than 1 cell. Similarly dying cells have greatly decreased or no metabolism which can be measured by these assays.

MTT reagent forms water-insoluble colored product which dictates an additional solubilization step necessary to get the final readout in the MTT assay. Key difference between MTT, and the more advanced reagent WST-1 is that WST-1 doesn’t require solubilization step. This is not only convenient but also valuable feature of WST-1 that decreases variability from the additional protocol steps. XTT reagent forms soluble product, but does require preliminary preparation of the labeling mixture. So our preference is always towards the ready-to-use WST-1 reagent which combines simple protocol with decreased assay variability due to the decreased number of steps.

Figure 3. Principle of WST-1 assay.

For one of our clients we have used WST-1 assay to test of plastics designed for cell growth support. Cells were plated on the test surfaces and their prolifersation was followed by WST-1 assay. Surfaces that gave the highest values for WST-1 assay in the pre-screening were shown later as the most appropriate for cell growth support in the battery of complementary tests.

WST-1 test hands-0n

1. Culture cells in 96-well plate in 100 ul of growth medium. Aim for 10,000-50,000 cells /well for optimal results, but as little as 500-1,000 cells can be detected using WST-1 reagent. Note: Save several wells with 100 ul growth medium but no cells for blank control – WST-1 reagent gives quite a bit of a background with traditional growth medium containing 10% FBS which can be accounted for using the blank wells.
2. Add 10 ul of WST-1 reagent to each well. Note: WST-1 is quite stable and can be stored for 1 month at 4C. For longer storage -20C or -70C would be a safer bet.
3. Incubate cells at 37C in cell culture incubator for 0.5-4 hours. Note: or until the highest value wells reach OD of ~2.0 at 440 nm
4. Shake plates well for 60 seconds to disperse colored formazan product throughout the medium.
5. Measure OD at 440 nm (anywhere between 420-470 nm would work) using wells with WST-1 and full growth media but no cells as blank.

OD would be proportional to cell number or cell proliferation depending on the experimental goal.

Finally, another useful dye that can be used to assess cell viability is resazurin (sold as Alamar Blue by Trek Diagnostic Systems, Vybrant by Molecular Probes and UptiBlue by Interchim). The deal with resazurin is that its products are relatively stable and non-toxic. As a result, you can add resazurin to cell culture medium and follow cell growth dynamics for 24-72 hours, although cell growth becomes progressively biased by resazurin degradation products as well as further degradation of resazurin colored products into non-colored by-products (3).

As a fun side note, resazurin was used in agriculture for 60+ years in assays to measure bacterial activity in milk or test a sperm quality. Then in 1990s Trek Diagnostic Systems developed and validated resazurin for mammalian cell proliferation assessment under Alamar Blue trademark while keeping its identity secret. In a dramatic exposure, Astra Zeneca team after doing some detective work with NMR and MS revealed “…the identity of Alamar Blue as resazurin” (3). Well, good for all of us, but probably folks at Trek Diagnostc Systems were less than happy by the unmasking of their flagship product. For the record, my personal preference is still for the original Trek product at least for experimental consistency sake.

Summary:

ATP levels and mitochondrial respiration are the most widely used surrogate markers to measure cell viability. These assays are amenable to high-throughput screening approaches but don’t allow discrimination of cell death mode. Decreased intracellular ATP or mitochondrial metabolism may result from non-lethal perturbations to cell growth and are ideal markers to measure senescence, proliferation, starvation, or contact inhibition. It should be noted that apoptosis or cell-mediated killing are active processes that in many cases may be accompanied by increased or stable cell metabolism.
With all that in mind, metabolic assays are perfect to obtain preliminary information about degree of cell growth inhibition. These results must be validated by secondary assays that rely on actual cell death markers, such as DNA fragmentation.

In the next post we will take a closer look at assays measuring bona fide cell death markers.

References:

1. Rita Hannah, Michael Beck, Richard Moravec, and Terry Riss, CellTiter-Glo Luminescent Cell Viability Assay, Promega Corporation  Cell Notes Issue 2 (2001) LINK TO FREE PDF
2. Roche Cytotoxicity Assay Special Interest Site LINK
3. O’Brien et al., Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity, Eur. J. Biochem. 267, 5422 (2000) LINK TO FREE PDF