How the ENO-30 Detects Nitrate and Nitrite

How the ENO-30 Detects Nitrate and Nitrite

An overview of the ENO-30, which can simultaneously detect nitrate and nitrite in biological and other samples.

The ENO-30 is a uniquely designed system that combines HPLC with a colorimetric assay optimized for fast and highly sensitive quantitative analysis of nitrate and nitrite. By utilizing the ENO-30, you can get accurate data in 10 minutes from a single injection. Combined with laboratory automation using an autosampler, you can run hundreds of samples overnight.

Let’s take a look inside to see how it works: The ENO-30 has an upper and lower unit. The lower unit contains the HPLC pumps optimized at just the right pumping speeds for the proper mixing of reagents.

Samples are injected with either a manual injector as shown or with an autosampler. Both nitrate and nitrite are first separated by retention time with an HPLC column. After separation, nitrate is reduced to nitrite. The system utilizes the Griess reaction, in which each nitrite forms an azo dye and is detected as the pink color develops and is measured by absorbance detection at 540 nm.

The design of the entire system has been optimized for highly reproducible results. It’s simple and easy to use. Here at Amuza, it’s important that instruments we provide work just as well for any given end-user as they do for us here at our own facility. That’s why we offer installation and training, as well as product support for all our products.

Thank you for watching. Feel free to contact us if you would like to learn more about running nitrate and nitrite samples.

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Obtain accurate fluid intake measurements

Obtain accurate fluid intake measurements

The Drinko Measurer is one of our most simple products, but an extremely useful one for gathering precise measurements of liquid consumption in animal studies.

What makes the Drinko Measurer unique is that it contains double ball bearings inside the nozzle of the sipper, preventing leakage of excess liquid. Furthermore, when force from a mouse or rat is applied to the tip of the sipper tube, a single drop of solution is applied.

Each tube also contains a clip that attaches to the sipper tube to the cage to stabilize the bottle to the cage. This prevents it from being knocked out of place by the mouse or rat. This clip can also be adjusted so you can adjust the length of the nozzle within the cage.

Additionally, all parts are autoclavable for easy cleaning.

The Drinko Measurer comes with several different sipper tube lengths 2.5, 3.5 and 4.5 inch as well as two different bottle sizes, 10 and 15ml. We also have new 15ml bottles with ml markings making it easier to visualize liquid consumption. However, we recommend weighing the bottles for the best results.

Now I’m going to tell you about several applications we recommend for the Drinko Measurer. The first is for drug delivery experiments.
For drugs that can be administered orally and easily mixed into a solvent, simply use the Drinko Measurer as a means for drug delivery rather than having to inject your experimental animals.
This is useful for drug dosing experiments and toxicology studies. Also, by combining two units per cage, you can measure drug-seeking preference with drugs of abuse.

The second application is for conditioned taste preference. Or conditioned taste aversion
Easily Measure preference or avoidance of a liquid by combining two Drinko Measurer systems and measuring liquid consumption.

Finally, we do carry 40 mL bottles which are useful for larger animals or for extended access studies. 

You can purchase units easily by navigating to our shop.

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Factors that Influence Experimental Outcomes and How to Overcome Them

Factors that Influence Experimental Outcomes and How to Overcome Them

Experimental outcomes can be influenced by a variety of factors, some of which can be controlled for. Minimizing confounding factors is crucial to gathering reliable and repeatable results.

One of the biggest issues in animal research today is the replicability of results. Too often animal study outcomes can not be repeated. This is not hard to believe given that the use of animals themselves provides inherent variability, even when all other factors are controlled for. Differences in the strain of animals used, as well as the age of the animals, time of day that experimental tests are administered, and how long the animals were handled prior to experimental testing are just some of the factors that can impact experimental outcomes.

While it is impossible to eliminate all external factors, animal-experimenter interactions can have a huge impact on results and should, therefore, try to be minimized as much as possible.

How can you mitigate animal-experimenter interactions?

At AMUZA, we offer a variety of automated behavioral tests that were designed specifically to improve the reliability and repeatability of behavioral assays.

For example, our Touch Panel operant training system is an automated operant chamber that utilizes photo beam sensors in the touch panel itself to improve the accuracy of responses from small rodents. The Touch Panel also includes software that enables users to design and run their own tasks with video tracking capabilities for automated data collection.

Even our standard mazes come with video tracking and automated data collection and analysis.

Touch Panel

Self Head-Restraining Platform

Furthermore, one of our other products, the Self Head-Restraining Platform, was designed to completely automate the head-fixation process in mice in order to streamline head-fixed behavioral assays.

In fact, the platform, originally developed by Dr. Andrea Benucci at RIKEN brain institute, was designed specifically to help overcome the reproducibility crisis.

Not only do our tools free up experimenter time and labor to focus on the actual science, they help remove unwanted experimenter bias by standardizing the experimental testing arena.

Even with automated behavioral tasks, however, it is still possible to introduce experimenter bias. This is why we also recommend that you perform rodent behavioral tests at roughly the same time each day, as well as handle experimental animals equally. Ideally, the same experimenter should be handling the animals each day. If this is not realistic, different experimenters should be counterbalanced across days, or across testing groups.

Also, if you plan to use different strains of mice or rats for your experiments, make sure to run behavioral tests across these different strains to account for any strain-specific differences.

Additionally, with our automated rodent behavior systems, we recommend that the motivation of the experimental animals to perform the task is consistent. If animals are food or water-deprived, weights should be taken daily initially and then weekly thereafter to ensure that test subjects are maintained at similar percentages of their free-feeding body weight.

Cleaning the testing chambers between use

All of our behavioral tests are made out of acrylic that is easy to clean as well as removable floors. Testing arenas should always be cleaned between experimental sessions to make sure the scent of the previous animal will not influence behavioral results.

For more detailed information about our automated behavioral tests connect with us today.

Benefits of using the ENO-30 for Nitrate and Nitrite Detection

Benefits of using the ENO-30 for Nitrate and Nitrite Detection

Today, I’d like to talk about a system capable of quantitative analysis of nitrate and nitrite from biological samples in minutes, called the ENO-30. The ENO-30 is a fast and highly sensitive system down to 0.1 picomoles.

Let me walk you through how easy it is to use. First, you get your sample ready for analysis. Sample prep is easy. In our manual, we’ve outlined a simple sample preparation protocol for tissue homogenate, blood, cell culture, urine, saliva, and microdialysis samples. Samples are injected into the ENO-30 with a manual injector or an autosampler for laboratory automation.

Once injected, nitrate and nitrite are separated by an HPLC separation column. Once separated, nitrate is reduced to nitrite, forming 2 separated nitrite compounds. Then, the separated nitrites are mixed with a Greiss reagent to form an azo dye, which is then detected by absorbance at 540 nm. In 10 minutes, you will be able to detect both nitrate and nitrite from a single injection.

The ENO-30 combines HPLC with colorimetric analysis. It’s like having a chemist and HPLC expert right on your benchtop. We’ve optimized the conditions to take away any guesswork and made the system straightforward and easy to use.

At Amuza, your success is our priority. It’s important that our products work for you just as well as they do for us here at our facility. When you acquire one of our machines, not only will it include installation and training, you’ll have access to our self-help support center as well as support from our knowledgeable support staff.

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How to Choose the Right Behavioral Test for Your Research

How to Choose the Right Behavioral Test for Your Research

New to Behavioral Research? Understanding how to choose the right behavioral test for your experiment is crucial.

There are several main categories of behavioral tests used to assess a variety of different brain functions and how they relate to behavior. In this post, we will describe these categories, the types of tests and what they measure.

Learning and Memory

There are a variety of mazes and operant tests that can be used to examine learning and memory in rodents. Below are some common examples, as well as what they are traditionally used to test.

Novel Object Recognition

This test is quite simple and can be performed inside the animal’s home cage. It is based on the tendency of rodents to spend more time exploring a novel object than a familiar one and requires the use of recognition memory.

Morris Water Maze

The Morris Water Maze is a water navigation task used to assess spatial learning and memory. In this task, animals must learn to find and remember where a hidden platform is that enables them to escape the water. Since rodents are averse to water, they are motivated to quickly find the hidden platform, which they cannot see because the water in the maze is opaque. Measuring response latency to the platform once the animal has learned where it is can be used to assess spatial working memory. This test can also be used to identify depression-like symptoms if the animal fails to show motivation to swim.

Barnes Maze

The Barnes Maze is also used to measure spatial learning and memory. The basic idea is to measure the ability of the experimental test subject to learn and remember the location of a target zone using distal cues located around the experimental testing arena.

The setup of the Barnes Maze consists of a circular surface with up to 20 around the outside of the circumference. Visual cues like-colored shapes or patterns are placed around the table for the animal to see. Under one of the holes is an “escape box” which the animal must learn to find using the cues. Rodents don’t like open spaces so they typically are very motivated to find the escape box.
Measuring the latency it takes for the animal to find the escape box is an indicator of spatial working memory.

T-Maze

The T-Maze spontaneous alternation test can be used to measure exploratory behavior. Rodents typically prefer to visit a new arm of the maze rather than a familiar one. The T-Maze can also be used to measure spatial working memory, by placing a reward at the end of one arm of the maze and then alternating the reward. The animal must learn that the arm that was previously not rewarded now is.

Y-Maze

The Y-Maze is very similar to the T-Maze with the exception that each of the arms is evenly spaced. The Y-Maze is thought to be slightly easier for rodents to learn compared to the T-Maze.
Interested in behavioral testing using several different maze types? Our Free Maze system at AMUZA gives you the opportunity to switch between up to eight different maze configurations. Learn more.

Fear Conditioning

Fear Conditioning (FC) is a type of associative learning task in which experimental test subjects learn that a previously neutral stimulus is associated with an aversive stimulus (foot shock). This learning is evidenced by anticipated freezing in response to the previously neutral cue even in the absence of the foot shock. With fear conditioning, animals learn to fear both the stimulus and the context that the stimulus is presented in. This test can be used to measure hippocampal-dependent contextual memory as well as fear processing in the amygdala.

Conditioned Place Preference

This is an operant test used to measure the motivational states that are connected to objects or experiences. You can measure both preference and avoidance by recording the amount of time the animal spends in the arena with the associated stimulus. This test is most commonly used to measure the rewarding and aversive effects of drugs. For these experiments, drugs are introduced in specific contexts, and then the animal is tested on how much time they spend in that particular context in the absence of the drug.

Sensorimotor Functioning

Open Field

The open-field test can be used to measure exploratory behavior and general locomotor activity in rodents and is a great test for measuring the toxicity of drugs in preclinical settings.

Accelerating Rotarod

In the accelerating rotarod test, the experimental test subject is placed on a rotating cylinder that is suspended in the air above the cage floor. Rodents will try to stay on the cylinder to avoid falling. The experimenter can measure the length of time that the animal stays on the rotarod. This is a great test to measure balance, coordination and motor planning and is effective for animal models of Parkinson’s disease and other neurodegenerative disorders.

Grip Strength Test

Also known as the forelimb grip strength test, in this test animals pull on a horizontal lever while the subjects are held by the tail and lowered towards the apparatus. Peak tension or the force applied to the bar just before the animal loses grip is applied. This test is useful for measuring and assessing deficits in motor function.

Addiction/Neuropsychiatric disorders

Startle Response and Pre-pulse Inhibition Test

Pre-pulse inhibition, also known as a reduction in startle response or PPI, is a phenomenon in which a weak stimulus (Pre-pulse) can suppress the startle response to a subsequent stronger startle stimulus (pulse). Impairments in PPI are thought to underlie impairments in sensorimotor gating, which is a common impairment seen in schizophrenia. Typically the way it works is that an animal is placed into a cylindrical chamber and a startling acoustic stimulus is played. The latency of the animal’s startle response to both the pre-pulse and the pulse can then be quantified.

 

Five-Choice Serial Reaction Time Task

The five-choice serial reaction time task, also known as the (5CSRTT) is commonly used to test attention and impulsivity in rodents. This task is typically carried out in an operant chamber, like our Touch Panel operant chamber, equipped with at least 5 holes. In this task, animals must correctly identify which of five holes has been illuminated via a nose poke. The time that the hole is illuminated can be shortened so that the animal must pay close attention in order to make the correct choice. Between trials, the experimental test subject must also inhibit responses to other holes until the next hole is illuminated. This task is quite useful for animal models of neuropsychiatric models like schizophrenia and autism.

Learn about all of the behavioral test options offered by AMUZA

5-minute Analysis of Dopamine and Serotonin in Brain Microdialysate

5-minute Analysis of Dopamine and Serotonin in Brain Microdialysate

In this video, we’ll show you how to detect and analyze dopamine and serotonin levels in brain microdialysis samples in 5 minutes. For this analysis, we use HPLC-ECD because it is a cost-effective way to get fast and ultra-sensitive data down to the femtomole range.

While HPLC-ECD is a common analysis method, careful consideration must be taken regarding analytical conditions, such as mobile phase preparation, column selection, and other factors. Ideally, we want:

  • Prompt elution to save time
  • Clear separation of the signal peaks from the solvent-front peaks
  • Fewer peaks around the signal peaks
  • A low noise level and a stable baseline

Optimizing these conditions can take time and resources. That’s why many labs require a dedicated person to handle HPLC. Thus, we’ve created an application to take out all the guesswork thereby maximizing the separation conditions for dopamine and serotonin.

For this application, we’re going to use the HTEC, which includes everything you need, integrated into one single unit. Let’s take a look inside:

  • Dual-piston pump with a unique algorithm to reduce noise without any pulse damper.
  • Degasser to remove small air bubbles for better pump performance.
  • Temperature control for consistent results.
  • Separation column uniquely selected for separation of DA and 5-HT.
  • Electrochemical detector cell with a 3 electrode DC potentiostat and an amperometric electrode.

The electrically active analyte is detected as it flows over the surface of a smooth electrode with an applied voltage.

We’ve optimized conditions to make it as easy as possible. Simply inject the sample using a manual injector or autosampler if you’re using laboratory automation. You can monitor signal response in real-time using the dedicated chromatography software. In 5 minutes you will have your results and you’re ready for the next sample. It’s highly sensitive down to 0.16 femtomoles.

Here’s an example of a chromatogram showing dopamine and serotonin.  

For more information on dopamine and serotonin analysis, contact us today!

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