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Micro-Volume Spectroscopy
Sponsored,vendor-submitted protocol   Sponsored by NanoDrop Technologies, Inc.    Published in BioTechniques Protocol Guide 2007 (p.51)

Absorbance and fluorescence measurements with minimal consumption of sample have become paramount as methods evolve, generating ever smaller amounts of material for analysis. Conventional measurements are impractical given the limited sample volumes produced by such techniques as laser-capture microdissection. The NanoDrop®ND-1000 Spectrophotometer and the NanoDrop®ND-3300 Fluorospectrometer can accurately measure a wide range of biomolecules in volumes as small as 1 microliter, using either absorbance or fluorescence analysis respectively. Both instruments employ a novel sample retention system that enables measurements without the use of traditional containment devices such as cuvettes or capillaries. The system uses fiber optic technology and inherent surface tension of liquid samples to hold a one microliter (1 ul) sample in place between two surfaces during the measurement cycle.

In order to make a measurement, 1 ul of sample is pipetted directly onto the lower (measurement) surface (Figure 1a). An upper pedestal automatically engages the sample, forming a liquid column of mechanically-controlled path length (Figure 1b).


Figure 1. (Click to enlarge)


Once the measurement is complete, the user simply blots both surfaces with a standard laboratory wipe to prepare for the next sample.

The NanoDrop®ND-1000 Spectrophotometer has a UV-Vis wavelength range of 220nm to 750nm, providing a versatile platform for absorbance analysis of various chromophores, including nucleic acids and proteins. By design, the ND-1000 spectrophotometer eliminates fixed containment devices, enabling automatic path length change from 1mm to 0.2mm during the same measurement cycle of a given sample. This allows for an unprecedented range of sample concentration to be assessed through absorbance spectroscopy (2ng/ul to 3700ng/ul for dsDNA), essentially eliminating the need to perform dilutions.

The NanoDrop®ND-3300 Fluorospectrometer performs full spectrum fluorescent analysis. Fluorophore excitation occurs from one of three LEDs: UV, blue, or white. The broad excitation range allows the measurement of a wide range of common fluorophores without the need for filter changes. The uniquely “clean optics” of the retention system and proprietary signal processing enable the unconventional use of the broad-spectrum, white LED. It can be used to excite several fluorophores, allowing for a multiple emission profile from a single sample.

The small sample volume requirement of the ND-3300 allows significantly scaled-down fluorescent reaction volumes (2-10ul), and even though the ND-3300 does not measure ultra low concentrations, it does lower the fluorescent detection limit of sample mass by more than an order of magnitude. For example, using the PicoGreen® dye, the ND-3300 can detect as little as 2pg dsDNA, while a cuvette or microplate PicoGreen® assay would need at least 25-50pg dsDNA for detection (Table A).


Table A. PicoGreen® Detection Limits (Click to enlarge)


NanoDrop Technologies’ novel retention system not only enables quality absorbance and fluorescence measurements with only a minute fraction of sample material needed to perform similar analysis on more traditional systems, but its ease-of-use provides a practical alternative for all spectroscopy measurements.

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The Bloody Fountain of Youth

12/09/2016
Elizabeth Harrett

Plasma transfusions mitigate signs of aging in mice—and early human studies show promise for the effects of young blood in people with Alzheimer’s Disease. Read more...


Parabiosis

Credit: Wikimedia

Whether through a fancy face cream, a new motorbike, or a ten-step exercise program, we are constantly sold the promise of remaining young. However, the biological processes underpinning getting older are much more serious than the cultural or aesthetic factors associated with reaching the twilight years. Age-related degenerative diseases are a serious concern, with dementia now the leading cause of death in the United Kingdom.

It is no surprise that many scientists study the precise mechanisms and markers that underpin aging, such as histone methylation and DNA repair proteins. But now, using an approach that sounds like a vampire folklore story, researchers have turned towards the very symbol of vitality and life itself: blood. They found that transfusions of young adult human blood can reverse symptoms of aging in older mice.

“Our mission is to expand an individual’s quality of life with the commensurate of the duration of their life,” said Joe McCracken, Vice President of Business Development for Alkahest, the company behind this study that was recently presented at the Society for Neuroscience (SfN) annual meeting in San Diego. “Our core technology is focused on the plasma proteome and understanding changes in the healthy proteome in age-related diseases such as Alzheimer’s.”

Back in 2014, the company published research looking exclusively at a mouse model of parabiosis, where elderly and young mice are joined to share a circulatory system (1). The researchers focused on the hippocampus, which is particularly vulnerable to aging.

Genome-wide microanalysis of these mice found improved synaptic plasticity–related transcriptional changes, dendritic spine density of mature neurons, and synaptic plasticity in aged parabionts. The molecular changes were not caused by the parabiosis procedure itself, suggesting that the improved plasticity was due to the blood exchange between old and young mice via the circulatory system.

To investigate the direct effect of blood, the researchers intravenously injected either young or aged murine plasma into older mice prior to cognitive testing, which involved using contextual fear conditioning and radial arm water maze paradigms. Performance improved in mice that received young blood in both contextual fear conditioning and spatial learning and memory tests.

The research presented at SfN built on this previous work, “We wanted to see the effects of human plasma, so we duplicated all studies in immunodeficient mice, which were used to prevent rejection,” said McCracken. “We found the results mimicked those published studying mouse plasma injections.”

The Alkahest research group examined the hippocampi of mice treated with old and young plasma, observing increased neuorogenesis (a process important for memory and learning) occurring in those injected with young plasma. They also used aptamer and antibody-based arrays and mass spectroscopy to identify potential therapeutic targets for age-related diseases, although Alkahest remains tight-lipped over what specific proteins they found.

Translating this research into clinical trials for humans has already began, “We’re just finishing the first study where we treated patients with mild to moderate Alzheimer’s disease with young human plasma. Plasma is presumably safe, but it’s never been used to treat Alzheimer’s, so with an abundance of caution, we started low dose infusions to test safety and did not see any adverse effects,” McCracken “By increasing the dose in frequency in our next study, we can continue to demonstrate safety and hopefully duplicate results found in mice.”

It is imperative to note that the work with clinical trials and human plasma being injected into mice has not yet been published in a peer-reviewed journal. However, this work is currently being prepared for publication. Indeed, McCracken stresses the importance of this work not being over-hyped, “We’d like to have the answers for a number of questions tomorrow, but it takes time to conduct rigorous science, and we really want to avoid creating unrealistic expectations.”

Reference

Villeda et al. (2014). Young blood reverses age-related impairments in cognitive function and synaptic plasticity in mice. Nature Medicine, 20: 659-663.

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