Introduction

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.

We are continuing our blog with a review of cell death assays for drug discovery. In this posting I will describe the most simple and useful assays to use in your project to measure cell death. All assays in our blog are available as a service from Alera Labs.

Introduction

As we have discussed in the first part of the series dedicated to cell death assays there are two major pathways for cell death: apoptosis and necrosis.

Early apoptosis leads to several specific changes, including:

1. Cell detachment from substrate
2. Phosphatidylserine exposure on the outer cell membrane
3. Nuclear condensation and DNA fragmentation
4. Generation of reactive oxygen species accompanied by lysosomal membrane permeabilization
5. Mitochondrial membrane permeabilization and activation of proteases associated with cell death (caspase and non-caspase proteases)
6. (in vivo apoptosis leads to formation of apoptotic bodies that are eaten by the neighboring phagocytes)

In cell culture, both late apoptosis and necrosis lead to:

1. A rupture of cell membrane with
2. The release of cell contents.

In the experimental set up it is also important to consider possible outcomes that are usually observed in case of assaying for cell death in the context of drug discovery:

1. No effect of the test compound on cell death.
2. Non-lethal effects such as effects on proliferation, senescence, starvation, mitochondrial respiration, and contact inhibition.
3. Cytotoxity - cell killing or non-lethal effect caused by chemical compounds or other cells. For example, cytotoxic  T-cells can form pores in the membrane of the target cell disrupting membrane integrity and causing cell lysis. This form of cell effects can be distinct from apoptosis and necrosis by virtue of using cellular pathways not involved in triggering of classic apoptosis or necrosis.
4. Apoptosis
5. Necrosis