Zone Preference in the Visual Water Maze
June 26, 2025
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Benchtop centrifuges are the equipment utilized in laboratories to separate and purify molecular mixtures in a liquid medium based on their density gradient. Centrifugation is a significant practice employed in biochemistry labs for analyzing and isolating cells, subcellular fractions, molecular complexes, and biological macromolecules like proteins, DNA, and RNA. Centrifuges are high-speed devices that require vacuum, gravitational acceleration, and centrifugal force to separate the desired molecules from the liquid mixtures without overheating the samples. Theodor Svedberg, a Nobel Laureate, developed the first analytical centrifuge in 1924 for monitoring the sedimentation process. Later, in the 1940s, Claude and his coworkers refined the centrifugation technique, making it the heart of biomedical and biological research for the upcoming decades (Wohlleben and Wendel, 2020). Currently, small-capacity benchtop centrifuges serve as a crucial tool in routine biomedical research.
The particles are uniformly distributed in a medium before centrifugation. The denser particles in the medium move towards the bottom, and the lighter particles move upwards upon centrifugation. After centrifugation, the top liquid fraction collected is called “supernatant,” The fraction that settles at the bottom is called “pellet.” An interface separates the supernatant and the pellet. The particle recovery in the pellet is the fraction of particles in the pellet post-centrifugation. This recovery depends on the particle density and diameter (Rikkert et al., 2018).
Analytical centrifugation is a separation technique that separates the samples based on their density by exposing them to centrifugal force. It is a method of studying a molecule’s hydrodynamic properties as it flows through the liquid medium. Analytical centrifuges are high-speed ultracentrifuges with optical systems for recording the sedimentation process. They characterize biological macromolecules based on their properties like molecular weight, density, diffusion, sedimentation coefficients, etc. Naturally, biological macromolecules possess random thermal motion unaffected by Earth’s gravitational field in an aqueous environment. However, isolated biomolecules show distinct sedimentation under high accelerations. A typical analytical centrifuge creates a centrifugal force of up to 200000xg. Analytical centrifuges work on the principle that molecules with higher molecular weight and density move more quickly and settle down faster than the smaller molecules with low density.
There are two main types of analytical centrifugation experiments: (1) sedimentation velocity (2) sedimentation equilibrium experiments. In sedimentation velocity experiments, a change in concentration distribution in the centrifuge cell at a high-speed rotor is recorded. In contrast, the steady-state concentration distribution is achieved in sedimentation equilibrium experiments. The hydrodynamic properties of the system are defined by their sedimentation coefficients. Three types of optical systems for analytical centrifuges are available, i.e., absorbance, interference, and fluorescence, that facilitate the selective and precise recording of sedimentation in real-time (Ohlendieck and Harding, 2018).
Preparative centrifugation is a separation technique that separates submicroscopic particles. Unlike analytical centrifuges, preparative centrifuges cannot be used in analytical procedures. Preparative centrifuges can process large sample quantities. There are two types of preparative centrifugation.
Differential centrifugation separates the biological particles of different sizes and densities based on the difference in their sedimentation rates. It involves a gradual application in the centrifugal field to divide crude tissue homogenates containing membrane vesicles, organelles, and structural fragments into different fractions. Differential centrifugation allows the initial sedimentation of the homogenate based on their density and relative size. The larger particles are sedimented in the initial centrifugation steps, leaving only smaller particles in the supernatant (Livshits et al., 2015). The cellular debris pellets can be homogenized repeatedly to increase the yield of membrane structures and protein aggregates. The largest particles sediment at the bottom of the centrifuge tube forming a pellet, whereas the smaller particles remain suspended in the supernatant.
Density gradient centrifugation is a technique in which the macromolecules move through a density gradient until they find a density equal to their own. This method is used for separating molecules of similar sizes yet different densities. For instance, cesium chloride centrifugation is employed for the plasmid and DNA isolation, and sodium bromide or sodium iodide are used for lipoprotein fractionation, and DNA/DNA banding. The sample and the gradient-forming solution are loaded in the centrifuge tube and rotated in a centrifuge. The centrifugal force distributes the cesium salts forming a density gradient from top to bottom. The sample molecule moves to the region that equals its own density. The two types of density gradient centrifugation include rate-zonal and isopycnic centrifugation.
Different types of benchtop centrifuges are listed below.
Microcentrifuges are the benchtop equipment suitable for processing low-volume sample tubes, with a small footprint and capacity to process up to 48 microtubes. They can provide a speed of approximately 6000rpm and process sample volumes of up to 2ml.
Mini centrifuges occupy even less space as compared to other benchtop centrifuges. They can process a maximum of eight tubes and have a maximum speed of 6000rpm. While such centrifuges are well-suited for research labs with less space, they might not be the best choice for high output laboratories.
Plate centrifuges are mainly used in PCR labs. These centrifuges ensure that all reagents are at the bottom of the wells for accurate concentrations and precise results. Plate centrifuges allow a horizontal spin at a maximum of 400xg speed. These benchtop centrifuges have a peculiar “wing-out rotor design” to prevent spillage.
The researchers use refrigerated centrifuges for the temperature-sensitive samples as even a mild change in temperature can ruin the samples. These are almost similar in design to their non-refrigerated benchtop counterparts. However, they allow temperature control within -10oC to 40oC.
Depending on the use in low-speed, high-speed, or ultra-centrifuge, different types of centrifugal forces are experienced by rotors. According to their use, the rotors can be made of different materials. For instance, low-speed rotors are made of brass or steel, whereas high-speed rotors are made of titanium, aluminum, or fiber-reinforced composites. The types of rotors mainly used in benchtop centrifuges include fixed-angle rotors, swinging bucket rotors, and vertical tube rotors.
Fixed angle rotors are used to separate biomolecules where the sedimentation rates significantly vary, such as nuclear, mitochondrial, and microsomal separation. For isopycnic separation, centrifugation is continued until the sample particles reach their lowest isopycnic gradient. It means that the sample has reached a position where the sedimentation rate is zero. Centrifugation tubes are held at a fixed angle ranging from 14o to 40o to the vertical, and the particles move radially outwards. Since the centrifugal field is exerted at an angle, they need to travel a short distance to reach their isopycnic position. They are used for differential centrifugation.
The tubes are held parallel to the rotational axis in vertical rotors and secured in the rotor cavities by screws, washers, and plugs. The samples are collected across the tube’s diameter instead of its length. Therefore, isopycnic separation time is significantly shorter. The tubes are held at an angle of 7° to 10°. They are usually used for density gradient centrifugation.
In-swinging bucket rotors hinge pins or crossbars are used to attach rotor buckets. The buckets are hung vertically, and when accelerated initially, the buckets swing out horizontally.
Benchtop centrifuges work on the sedimentation principle, i.e., substances separate according to their density under the influence of Earth’s gravitational force ‘g’ (g = 9.81ms-2). The sedimentation rate increases when these samples undergo acceleration in a centrifugal field (G > 9.81ms-2). The relative gravitational field is often expressed as a multiple of gravitational acceleration. Underlying points must be considered while working with benchtop centrifuges.
The frictional force experienced by a biological medium in a viscous medium is in a direction opposite to that of sedimentation. It is equal to the product of the particle’s velocity and frictional coefficient. As described earlier, the centrifugal field is related to Earth’s gravitational field. The relative centrifugal field (RCF) is the ratio of centrifugal force at a specified radius and speed to the standard gravitational acceleration (Ohlendieck and Harding, 2018). The RCF can be calculated from the formula presented below:
Insert the sample tube in one of the portals. Based on the number of samples you are centrifuging, add tubes filled with water for balancing. Secure the lid and select desired settings. Press the “start” button and wait for the centrifuge cycle to complete. Once the centrifuge stops spinning, take out the sample and the balances. Notice that the sample is differentiated into two parts, i.e., supernatant and pellet. Analyze the sample as per your sample requirement.
Conduct Science presents the best range of benchtop centrifuges with an easy-to-use ergonomic design that complements your laboratory requirements. These benchtop equipment are made of durable and high-quality material and bring efficiency and consistency in experiments. Different benchtop models possess two types of rotors: (1) out-swung rotors (2) fixed-angle rotors. These centrifuges have a maximum 250 ml instrument capacity and can provide a variable speed of up to 6000rpm. Moreover, they can give a maximum RCF of 20,000xg. Other features of these instruments include continuous hold-spin function, rubber suction feet for grip at the benchtop, and last-spin memory to record the last centrifuge experiment. The researchers can also buy microcentrifuges or ultracentrifuges with a compact design best suited for their workspace.
Benchtop centrifuges have various applications in microbiology, biochemistry, and biomedical research. Differential centrifugation and density gradient centrifugation isolate microsomal fractions from muscle homogenates, isolate highly purified sarcolemmal vesicles, and sub fractionation liver mitochondrial membrane systems. A few applications of benchtop centrifuges are given below.
Platelet Rich Fibrin (PRF) Production
Second-generation platelet-rich fibrin (PRF) is now used as a “therapeutic strategy for promoting implant healing and bone and soft tissue integration” in dental implants. Platelet-rich fibrin (PRF) or leukocyte and platelet-rich fibrin (L-PRF) are generally obtained from patients’ blood and centrifuged at 700xg RCF for 12 minutes without additives. Feng et al. (2020) studied the antibacterial properties of PRF prepared by horizontal centrifugation against E. coli and S. aureus. The researchers reviewed that PRF’s horizontal centrifugation yields better layer separation by minimizing cell accumulation at distal ends of centrifugal tubes. The swung-out buckets used in horizontal centrifugation produce a completely horizontal tube. It causes substantial differences between RCF-min and RCF-max due to the difference in minimum and maximum tube radii. They reviewed that horizontal centrifugation could increase the yield fourfold compared to fixed-angle centrifugation. Therefore, they aimed to compare the antimicrobial effects of horizontal centrifugation (H-PRF) and L-PRF were produced on a fixed angle rotor against S. aureus and E. coli and determined whether these antibacterial effects were correlated with immune cell number. In the case of L-PRF preparation, the experimenters centrifuged the samples using fixed-angle rotors and observed an angular red blood layer formed within the centrifuge tube. On the contrary, in the case of horizontal centrifugation, the researchers observed a horizontal division between red blood cells and platelet-rich fibrin. Since cell content in each PRF layer is different, and H-PRF can harbor up to four times more leukocytes than L-PRF, the researchers divided liquid state PRF into five equal portions following centrifugation. From the CFU examination and flow-cytometric analysis of these samples, the experimenters concluded that solid PRF exhibits better antimicrobial activity than liquid PRF.
Isolation of Biopsy-based Tumor Biomarkers
Cancer patients’ blood contains numerous biomarkers, including circulating tumor cells (CTC), circulating cell-free DNA (ccfDNA), and tumor-educated platelets (TEPs). As these biomarkers differ in size and density, the lab personnel usually centrifuge blood to isolate or concentrate the biomarker of interest. Rikkert et al. (2018) devised Stokes’s law-based model to estimate the effect of centrifugation on biomarker purity and then applied this model to predict biomarker behavior during centrifugation. According to Stoke’s law, the acceleration due to gravity depends on the distance from the rotational axis in a centrifuge. This gravitational acceleration is given by:
g = Rω2
where R is the distance to the axis of rotation and ω is the angular velocity. Stoke’s equation is usually used in the case of the swing-out rotor. They diluted polystyrene beads in phosphate buffer saline (PBS) or blood plasma to validate the suggested model. They subjected the sample to centrifugation at 300g for 20 minutes, 2700g for 22 minutes, or 60 minutes at 15,800g. They also measured bead concentration by flow cytometry before and after centrifugation. The experimenters concluded that Stokes law could be used to predict biomarker behavior in blood. They also concluded that centrifugation alone could not isolate a single biomarker since other coisolated biomarkers remained present in significant amounts.
Benchtop centrifuges are a perfect fit for the research laboratories with less benchtop space due to their compact design. These centrifuges are easy-to-use and offer fast start-up and shut down. They are fully automated hence reducing the possibility of experimental failure due to human error. Moreover, their enclosed design keeps the specimens uncontaminated. However, a potential disadvantage of centrifuges is their higher energy demand (Brandt et al., 2017).
Wohlleben, W., Coleman, V. A., & Gilliland, D. (2020). Analytical centrifugation. In Characterization of Nanoparticles (pp. 225-247). Elsevier.
Livshits, M. A., Khomyakova, E., Evtushenko, E. G., Lazarev, V. N., Kulemin, N. A., Semina, S. E., … & Govorun, V. M. (2015). Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol. Scientific reports, 5(1), 1-14.
Brandt, M. J., Johnson, K. M., Elphinston, A. J., & Ratnayaka, D. D. (2017). Chapter 13—Energy Use, Sustainability and Waste Treatment. Twort’s Water Supply, 7th Ed.; Brandt, MJ, Johnson, KM, Elphinston, AJ, Ratnayaka, DD, Eds, 553-580.
Rikkert, L. G., Van Der Pol, E., Van Leeuwen, T. G., Nieuwland, R., & Coumans, F. A. (2018). Centrifugation affects the purity of liquid biopsy‐based tumor biomarkers. Cytometry Part A, 93(12), 1207-1212.
Feng, M., Wang, Y., Zhang, P., Zhao, Q., Yu, S., Shen, K., … & Zhang, Y. (2020). Antibacterial effects of platelet-rich fibrin produced by horizontal centrifugation. International journal of oral science, 12(1), 1-8.
Ohlendieck, K., & Harding, S. E. (2018). Centrifugation and Ultracentrifugation. Wilson and Walker’s Principles and Techniques of Biochemistry and Molecular Biology.
In behavioral neuroscience, the Open Field Test (OFT) remains one of the most widely used assays to evaluate rodent models of affect, cognition, and motivation. It provides a non-invasive framework for examining how animals respond to novelty, stress, and pharmacological or environmental manipulations. Among the test’s core metrics, the percentage of time spent in the center zone offers a uniquely normalized and sensitive measure of an animal’s emotional reactivity and willingness to engage with a potentially risky environment.
This metric is calculated as the proportion of time spent in the central area of the arena—typically the inner 25%—relative to the entire session duration. By normalizing this value, researchers gain a behaviorally informative variable that is resilient to fluctuations in session length or overall movement levels. This makes it especially valuable in comparative analyses, longitudinal monitoring, and cross-model validation.
Unlike raw center duration, which can be affected by trial design inconsistencies, the percentage-based measure enables clearer comparisons across animals, treatments, and conditions. It plays a key role in identifying trait anxiety, avoidance behavior, risk-taking tendencies, and environmental adaptation, making it indispensable in both basic and translational research contexts.
Whereas simple center duration provides absolute time, the percentage-based metric introduces greater interpretability and reproducibility, especially when comparing different animal models, treatment conditions, or experimental setups. It is particularly effective for quantifying avoidance behaviors, risk assessment strategies, and trait anxiety profiles in both acute and longitudinal designs.
This metric reflects the relative amount of time an animal chooses to spend in the open, exposed portion of the arena—typically defined as the inner 25% of a square or circular enclosure. Because rodents innately prefer the periphery (thigmotaxis), time in the center is inversely associated with anxiety-like behavior. As such, this percentage is considered a sensitive, normalized index of:
Critically, because this metric is normalized by session duration, it accommodates variability in activity levels or testing conditions. This makes it especially suitable for comparing across individuals, treatment groups, or timepoints in longitudinal studies.
A high percentage of center time indicates reduced anxiety, increased novelty-seeking, or pharmacological modulation (e.g., anxiolysis). Conversely, a low percentage suggests emotional inhibition, behavioral avoidance, or contextual hypervigilance. reduced anxiety, increased novelty-seeking, or pharmacological modulation (e.g., anxiolysis). Conversely, a low percentage suggests emotional inhibition, behavioral avoidance, or contextual hypervigilance.
The percentage of center time is one of the most direct, unconditioned readouts of anxiety-like behavior in rodents. It is frequently reduced in models of PTSD, chronic stress, or early-life adversity, where animals exhibit persistent avoidance of the center due to heightened emotional reactivity. This metric can also distinguish between acute anxiety responses and enduring trait anxiety, especially in longitudinal or developmental studies. Its normalized nature makes it ideal for comparing across cohorts with variable locomotor profiles, helping researchers detect true affective changes rather than activity-based confounds.
Rodents that spend more time in the center zone typically exhibit broader and more flexible exploration strategies. This behavior reflects not only reduced anxiety but also cognitive engagement and environmental curiosity. High center percentage is associated with robust spatial learning, attentional scanning, and memory encoding functions, supported by coordinated activation in the prefrontal cortex, hippocampus, and basal forebrain. In contrast, reduced center engagement may signal spatial rigidity, attentional narrowing, or cognitive withdrawal, particularly in models of neurodegeneration or aging.
The open field test remains one of the most widely accepted platforms for testing anxiolytic and psychotropic drugs. The percentage of center time reliably increases following administration of anxiolytic agents such as benzodiazepines, SSRIs, and GABA-A receptor agonists. This metric serves as a sensitive and reproducible endpoint in preclinical dose-finding studies, mechanistic pharmacology, and compound screening pipelines. It also aids in differentiating true anxiolytic effects from sedation or motor suppression by integrating with other behavioral parameters like distance traveled and entry count (Prut & Belzung, 2003).
Sex-based differences in emotional regulation often manifest in open field behavior, with female rodents generally exhibiting higher variability in center zone metrics due to hormonal cycling. For example, estrogen has been shown to facilitate exploratory behavior and increase center occupancy, while progesterone and stress-induced corticosterone often reduce it. Studies involving gonadectomy, hormone replacement, or sex-specific genetic knockouts use this metric to quantify the impact of endocrine factors on anxiety and exploratory behavior. As such, it remains a vital tool for dissecting sex-dependent neurobehavioral dynamics.
The percentage of center time is one of the most direct, unconditioned readouts of anxiety-like behavior in rodents. It is frequently reduced in models of PTSD, chronic stress, or early-life adversity. Because it is normalized, this metric is especially helpful for distinguishing between genuine avoidance and low general activity.
Environmental Control: Uniformity in environmental conditions is essential. Lighting should be evenly diffused to avoid shadow bias, and noise should be minimized to prevent stress-induced variability. The arena must be cleaned between trials using odor-neutral solutions to eliminate scent trails or pheromone cues that may affect zone preference. Any variation in these conditions can introduce systematic bias in center zone behavior. Use consistent definitions of the center zone (commonly 25% of total area) to allow valid comparisons. Software-based segmentation enhances spatial precision.
Evaluating how center time evolves across the duration of a session—divided into early, middle, and late thirds—provides insight into behavioral transitions and adaptive responses. Animals may begin by avoiding the center, only to gradually increase center time as they habituate to the environment. Conversely, persistently low center time across the session can signal prolonged anxiety, fear generalization, or a trait-like avoidance phenotype.
To validate the significance of center time percentage, it should be examined alongside results from other anxiety-related tests such as the Elevated Plus Maze, Light-Dark Box, or Novelty Suppressed Feeding. Concordance across paradigms supports the reliability of center time as a trait marker, while discordance may indicate task-specific reactivity or behavioral dissociation.
When paired with high-resolution scoring of behavioral events such as rearing, grooming, defecation, or immobility, center time offers a richer view of the animal’s internal state. For example, an animal that spends substantial time in the center while grooming may be coping with mild stress, while another that remains immobile in the periphery may be experiencing more severe anxiety. Microstructure analysis aids in decoding the complexity behind spatial behavior.
Animals naturally vary in their exploratory style. By analyzing percentage of center time across subjects, researchers can identify behavioral subgroups—such as consistently bold individuals who frequently explore the center versus cautious animals that remain along the periphery. These classifications can be used to examine predictors of drug response, resilience to stress, or vulnerability to neuropsychiatric disorders.
In studies with large cohorts or multiple behavioral variables, machine learning techniques such as hierarchical clustering or principal component analysis can incorporate center time percentage to discover novel phenotypic groupings. These data-driven approaches help uncover latent dimensions of behavior that may not be visible through univariate analyses alone.
Total locomotion helps contextualize center time. Low percentage values in animals with minimal movement may reflect sedation or fatigue, while similar values in high-mobility subjects suggest deliberate avoidance. This metric helps distinguish emotional versus motor causes of low center engagement.
This measure indicates how often the animal initiates exploration of the center zone. When combined with percentage of time, it differentiates between frequent but brief visits (indicative of anxiety or impulsivity) versus fewer but sustained center engagements (suggesting comfort and behavioral confidence).
The delay before the first center entry reflects initial threat appraisal. Longer latencies may be associated with heightened fear or low motivation, while shorter latencies are typically linked to exploratory drive or low anxiety.
Time spent hugging the walls offers a spatial counterbalance to center metrics. High thigmotaxis and low center time jointly support an interpretation of strong avoidance behavior. This inverse relationship helps triangulate affective and motivational states.
By expressing center zone activity as a proportion of total trial time, researchers gain a metric that is resistant to session variability and more readily comparable across time, treatment, and model conditions. This normalized measure enhances reproducibility and statistical power, particularly in multi-cohort or cross-laboratory designs.
For experimental designs aimed at assessing anxiety, exploratory strategy, or affective state, the percentage of time spent in the center offers one of the most robust and interpretable measures available in the Open Field Test.
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