Measurement of Reactive Oxygen Species (ROS) in the Microplate Format

Reactive Oxygen Species (ROS) is a phrase used to describe a number of reactive molecules and free radicals derived from molecular oxygen. Reactive oxygen species were originally thought to be only released by phagocytic cells as part of their role in host defense. Recent work has demonstrated that ROS have a role in cell signaling, including; apoptosis; gene expression; and the activation of cell signaling cascades.

The measurement of reactive oxygen species is dependent on the analytic target. At the cellular level specific reactive oxygen species can be individually assessed from tissue culture, while at the whole animal level typically the effects of oxidative stress are measured from blood product (e.g. serum or plasma) or from urine samples. Depending on the ROS species of interest, measurements can be made using any one of the three different read modalities: absorbance; fluorescence; and luminescence. For example, glutathione, the most significant non enzymatic oxidant defense mechanisms can be measured using absorbance [1] or luminescence [2]. Likewise lipid peroxidation can be assessed by the detection of detection of thiobarbituric acid (TBA) reactive compounds such as malondialdehyde in microplates colorimetrically at 532 nm or by fluorescence using a 530 nm excitation wavelength and a 550 nm emission wavelength [3].

Mitogenic stimulation of cells has been shown to result in an increase in hydrogen peroxide (H2O2), which can be detected by the production of fluorescence from dichlorofluorescein (DCF) [4] or Peroxy Green 1 (PG-1) [5]. These compounds are non-fluorescent until they are oxidized within the cell by reactive oxygen species.

The multimode readers from BioTek, such as the Synergy 2, Synergy 4 and Synergy Mx are ideal platforms for quantitation of these compounds and many others associated reactive oxygen species. In addition to the detection readers available from BioTek, the Precision pipettor and MicroFill or MicroFlo dispensers can be used to pipette samples or dispense reagents respectively.

Do you have any interest in measuring reactive oxygen species in microplates? Do you currently measure ROS using flow cytometry or by microscopy? What indicator dye do you use?


References

1. Baker, MA, G.J. Cerniglia, and A. Zaman. (1990) Microtiter plate Assay for the measurement of glutathione and glutathione disulfide in large numbers of Biological Samples. Anal. Biochem., 190:360-365
2. Promega GSH-Glo Glutathione Assay Technical Bulletin, TB369, Promega Corporation, Madison, WI.
3. Pryor, W.A., J.P. Stanley, and E. Blair. (1976) Autoxidation of polyunsaturated fatty acids: II. A Suggested mechanism for the Formation of TBA-reactive materials from prostaglandin-like Endoperoxides. Lipids, 11:370-379.
4. Tarpley, M.M., D.A. Wink, and M.B. Grisham (2004) Methods for detection of reactive Metabolites of Oxygen and Nitrogen: in vitro and in vivo considerations. Am . J. Physiol Regul Integr Comp Physiol. 286:R431-R444.
5. Miller, E.V., O. Tulyathan, E.Y. Isacoff, and C.J. Chang (2007). Molecular imaging of hydrogen peroxide produced for cell signaling. Nature Chemical Biology, 3(5):263-267.

Automation of Luminescence-based CYP Inhibition Assays in Drug Discovery

Most small molecule drugs are metabolized predominantly in the liver by cytochrome P450 (CYP) enzymes, particularly CYP isoforms 3A4, 2C9 and 2D6. It is important to assess metabolism for appropriate dosing, but also for establishing metabolism-related drug-drug interactions where one drug may affect the metabolism of another, leading to possible toxic effects. The ability to determine the effects that lead compounds have on these enzymes is an important part of today’s drug discovery routine. One desired component of this process is the ability to profile compounds against multiple CYP450 enzymes using the same basic procedure. The second is easy to use, yet dependable instrumentation that can be used to dilute compounds, as well as dispense assay reagents, that will create accurate titration curves.

To demonstrate automated CYP profiling, we used BioTek’s Precision™ XS Automated Pipetting System and Synergy™ MX Monochromator-Based Multi-Mode Microplate Reader, along with Promega’s P450-Glo™ 2C9, 3A4, and 2D6 Screening Systems. The Precision was used to perform compound titrations, as well as dispense all assay components to the wells of the assay plates. The Synergy was used to detect the luminescent signal from each well. Titrations were carried out in 96-well format, with a 20 µL transfer volume. 5 µL of compound was transferred in quadruplicate to the assay plate, followed by 5 µL of enzyme/substrate mix. 10 µL of NADPH Regeneration System was then dispensed to start the reaction. After the appropriate incubation time, 20 µL of Luciferin Detection Reagent was added to the plate.

Z’-Factor assays were run to validate the automated methods. Compounds were then profiled against all three CYP450 isoforms on the same 384-well assay plate to demonstrate the ease of the combined procedure. Agreement of IC50 values for control compounds with established literature values confirms that the combination of robotics, detection, and chemistry creates an ideal solution for automated cytochrome P450 profiling of lead compounds in drug discovery campaigns.

Where do you see cytochrome P450 profiling demands going in the future? Will there be an increased need for cell-based CYP450 assays? What type of throughput do you feel is essential for automated CYP450 profiling?

Table: Compound IC50 values using P450-Glo™ and Control Assays

Graph: Compounds Inhibition Curves for CYP3A4 Isoform

Drug Discovery & Development Week

Just last week I attended the Annual Drug Discovery & Development Conference held at the World Trade Center/Seaport Hotel in Boston, Massachusetts. Armed with an Exhibitors Pass I was relegated to the Exhibition Floor and immediately noticed a dramatic drop in both exhibitor and conferee attendance. In just two short years the exhibition hall went from over 200 companies to 64. From an attendees standpoint, this is not a good sign as product exhibitions are a great opportunity for conference attendees to see the latest in reagent and instrument technologies. For BioTek, the number of leads was also down considerably, but the ones we obtained were of high value. This leads one to think about the future of conferences. Will participants only attend one conference per year? Will more "specialized" or "applications" focused meetings be the wave of the future, or will "virtual" conferences become the norm? I'd be interested in hearing any comments on the topic.

Of our competition, only BMG, Beckman Coulter and TiterTek/Berthold DS were exhibiting products. One company that caught my interest was Formulatrix, who demonstrated a unique and interesting technology to dispense down to 200 nL volumes independently into a microplate. The product seemed to be at the prototype stage but did generate considerable interest.

Next year IBC plans to move the conference to San Diego. While I wish them well, I don't expect a recovery. I must add that a trip to the north section of Boston, known as "Little Italy" was still the treat it has always been and would recommend the dining and strolling down the streets to anyone.

Low-volume Quantification of Nucleic Acids using a Monochromator-based Microplate Spectrophotometer

The quantification of nucleic acids is a necessary procedure after isolation from real samples such as tissues, cells or body fluids. Downstream applications include PCR, RT-PCR, sequencing,restriction digestions and ligations. All these applications involve enzymatic reactions where efficiency is dependent on the relative concentrations of nucleic acid, enzyme and other reactants, hence the need for quantification. Spectrophotometery is a very popular method for nucleic acid quantification as it is a simple, accurate and non-destructive method for the measurement of nucleic acid concentrations although for standard 1 cm path length cuvettes, dilution of the sample is typically required.The Epoch™ Multi-Volume Spectrophotometer System is a flexible instrument that provides microvolume quantification of nucleic acids (dsDNA, ssDNA, oligonuleotides) from sub-ng/uL to 4,000 ng/uL without the need for sample dilution. Microanalysis can be performed with as little as 2 uL on 1 – 16 samples simultaneously. The Take3 microplate also allows for 1 cm path length quantification using BioTek’s BioCell accessory or a standard spectrophotometer cuvette. In addition, the Take3 Multi-Volume Plate can be replaced by standard 6 – 384-well microplates for many other applications.

The accuracy of the microvolume quantification of dsDNA is demonstrated in the graph below where standard samples from herring sperm has been measured using undiluted 2 mL samples in the Take3 plate (average of 16 measurements from each of the microwells of the Take3 microplate) and compared to 1 cm pathlength (diluted samples).

It is evident that there is excellent correlation between Take3 microvolume determinations and gold standard cuvette-based measurements.

How would you use the plate to take advantage of the 16 well locations? What applications would benefit most from the ability to read low-volume samples, microplates and cuvettes from the same technology?