Chemotaxis assays are experimental tools for evaluation of chemotactic ability of prokaryotic or eukaryotic cells. A wide variety of techniques have been developed. Some techniques are qualitative - allowing an investigator to approximately determine a cell's chemotactic affinity for an analyte - while others are quantitative, allowing a precise measurement of this affinity.
In general, the most important requisite is to calibrate the incubation time of the assay both to the model cell and the ligand to be evaluated. Too short incubation time results in no cells in the sample, while too long time perturbs the concentration gradients and measures more chemokinetic than chemotactic responses.
The most commonly used techniques are grouped into two main groups:
This way of evaluation deals with agar-agar or gelatine containing semi-solid layers made prior to the experiment. Small wells are cut into the layer and filled with cells and the test substance. Cells can migrate towards the chemical gradient in the semi solid layer or under the layer as well. Some variations of the technique deal also with wells and parallel channels connected by a cut at the start of the experiment (PP-technique). Radial arrangement of PP-technique (3 or more channels) provides the possibility to compare chemotactic activity of different cell populations or study preference between ligands. [1]
Counting of cells: positive responder cells could be counted from the front of migrating cells, after staining or in native conditions in light microscope.
Chambers isolated by filters are proper tools for accurate determination of chemotactic behavior. The pioneer type of these chambers was constructed by Boyden. [2] The motile cells are placed into the upper chamber, while fluid containing the test substance is filled into the lower one. The size of the motile cells to be investigated determines the pore size of the filter; it is essential to choose a diameter which allows active transmigration. For modelling in vivo conditions, several protocols prefer coverage of filter with molecules of extracellular matrix (collagen, elastin etc.) Efficiency of the measurements was increased by development of multiwell chambers (e.g. NeuroProbe), where 24, 96, 384 samples are evaluated in parallel. Advantage of this variant is that several parallels are assayed in identical conditions.
In another setting, the chambers are connected side by side horizontally (Zigmond chamber) [3] or as concentric rings on a slide (Dunn chamber) [4] Concentration gradient develops on a narrow connecting bridge between the chambers and the number of migrating cells is also counted on the surface of the bridge by light microscope. In some cases the bridge between the two chambers is filled with agar and cells have to "glide" in this semisolid layer.
Some capillary techniques provide also a chamber like arrangement, however, there is no filter between the cells and the test substance. [5] Quantitative results are gained by the multiwell type of this probe using 4-8-12-channel pipettes. Accuracy of the pipette and increased number of the parallel running samples is the great advantage of this test. [6]
Counting of cells: positive responder cells are count from the lower chamber (long incubation time) or from the filter (short incubation time). For detection of cells general staining techniques (e.g. trypan blue) or special probes (e.g. mt-dehydrogenase detection with MTT assay) are used. Labelled (e.g. fluorochromes) cells are also used, in some assays cells get labelled during transmigration the filter.
Besides the above-mentioned two most commonly used family of techniques, a wide range of protocols were developed to measure chemotactic activity. Some of them are only qualitative, like aggregation tests, where small pieces of agar or filters are placed onto a slide and accumulation of cells around is measured.
In another semiquantitative technique, cells are overlaid the test substance and changes in opalescence of the originally cell-free compartment is recorded during the incubation time. [7]
The third frequently used qualitative technique is the T-maze and its adaptations for microplates. In the original version, a container drilled in a peg is filled with cells. Then the peg is twisted and the cells get contact with two other containers filled with different substances. The incubation is stopped by resetting the peg and the cell number is counted from the containers. [8]
Also, lately,[ when? ] microfluidic devices have been used more and more frequently to test quantitatively, and precisely, for chemotaxis. [9] [10] [11]
Agar, or agar-agar, is a jelly-like substance consisting of polysaccharides obtained from the cell walls of some species of red algae, primarily from ogonori (Gracilaria) and "tengusa" (Gelidiaceae).
Chemotaxis is the movement of an organism or entity in response to a chemical stimulus. Somatic cells, bacteria, and other single-cell or multicellular organisms direct their movements according to certain chemicals in their environment. This is important for bacteria to find food by swimming toward the highest concentration of food molecules, or to flee from poisons. In multicellular organisms, chemotaxis is critical to early development and development as well as in normal function and health. In addition, it has been recognized that mechanisms that allow chemotaxis in animals can be subverted during cancer metastasis. The aberrant chemotaxis of leukocytes and lymphocytes also contribute to inflammatory diseases such as atherosclerosis, asthma, and arthritis. Sub-cellular components, such as the polarity patch generated by mating yeast, may also display chemotactic behavior.
Microfluidics refers to the behavior, precise control, and manipulation of fluids that are geometrically constrained to a small scale at which surface forces dominate volumetric forces. It is a multidisciplinary field that involves engineering, physics, chemistry, biochemistry, nanotechnology, and biotechnology. It has practical applications in the design of systems that process low volumes of fluids to achieve multiplexing, automation, and high-throughput screening. Microfluidics emerged in the beginning of the 1980s and is used in the development of inkjet printheads, DNA chips, lab-on-a-chip technology, micro-propulsion, and micro-thermal technologies.
Virology is the scientific study of biological viruses. It is a subfield of microbiology that focuses on their detection, structure, classification and evolution, their methods of infection and exploitation of host cells for reproduction, their interaction with host organism physiology and immunity, the diseases they cause, the techniques to isolate and culture them, and their use in research and therapy.
An assay is an investigative (analytic) procedure in laboratory medicine, mining, pharmacology, environmental biology and molecular biology for qualitatively assessing or quantitatively measuring the presence, amount, or functional activity of a target entity. The measured entity is often called the analyte, the measurand, or the target of the assay. The analyte can be a drug, biochemical substance, chemical element or compound, or cell in an organism or organic sample. An assay usually aims to measure an analyte's intensive property and express it in the relevant measurement unit.
A microbiological culture, or microbial culture, is a method of multiplying microbial organisms by letting them reproduce in predetermined culture medium under controlled laboratory conditions. Microbial cultures are foundational and basic diagnostic methods used as a research tool in molecular biology.
A colony-forming unit is a unit used in microbiology. It estimates the number of bacteria or fungal cells in a sample which are viable, able to multiply via binary fission under the controlled conditions. Counting with colony-forming units requires culturing the microbes and counts only viable cells, in contrast with microscopic examination which counts all cells, living or dead. The visual appearance of a colony in a cell culture requires significant growth, and when counting colonies it is uncertain if the colony arose from one cell or a group of cells. Expressing results as colony-forming units reflects this uncertainty.
Chemokinesis is chemically prompted kinesis, a motile response of unicellular prokaryotic or eukaryotic organisms to chemicals that cause the cell to make some kind of change in their migratory/swimming behaviour. Changes involve an increase or decrease of speed, alterations of amplitude or frequency of motile character, or direction of migration. However, in contrast to chemotaxis, chemokinesis has a random, non-vectorial moiety, in general.
Due to the random character, techniques dedicated to evaluate chemokinesis are partly different from methods used in chemotaxis research. One of the most valuable ways to measure chemokinesis is computer-assisted checker-board analysis, which provides data about migration of identical cells, whereas, in Protozoa, techniques based on measurement of opalescence were also developed.
Chemotaxis receptors are expressed in the surface membrane with diverse dynamics, some of them have long-term characteristics as they are determined genetically, others have short-term moiety as their assembly is induced ad hoc in the presence of the ligand. The diverse feature of the chemotaxis receptors and ligands provides the possibility to select chemotactic responder cells with a simple chemotaxis assay. By chemotactic selection we can determine whether a still not characterized molecule acts via the long- or the short-term receptor pathway. Recent results proved that chemokines are working on long-term chemotaxis receptors, while vasoactive peptides act more on the short-term ones. Term chemotactic selection is also used to design a technique which separates eukaryotic or prokaryotic cells upon their chemotactic responsiveness to selector ligands.
Bio-MEMS is an abbreviation for biomedical microelectromechanical systems. Bio-MEMS have considerable overlap, and is sometimes considered synonymous, with lab-on-a-chip (LOC) and micro total analysis systems (μTAS). Bio-MEMS is typically more focused on mechanical parts and microfabrication technologies made suitable for biological applications. On the other hand, lab-on-a-chip is concerned with miniaturization and integration of laboratory processes and experiments into single chips. In this definition, lab-on-a-chip devices do not strictly have biological applications, although most do or are amenable to be adapted for biological purposes. Similarly, micro total analysis systems may not have biological applications in mind, and are usually dedicated to chemical analysis. A broad definition for bio-MEMS can be used to refer to the science and technology of operating at the microscale for biological and biomedical applications, which may or may not include any electronic or mechanical functions. The interdisciplinary nature of bio-MEMS combines material sciences, clinical sciences, medicine, surgery, electrical engineering, mechanical engineering, optical engineering, chemical engineering, and biomedical engineering. Some of its major applications include genomics, proteomics, molecular diagnostics, point-of-care diagnostics, tissue engineering, single cell analysis and implantable microdevices.
Sperm guidance is the process by which sperm cells (spermatozoa) are directed to the oocyte (egg) for the aim of fertilization. In the case of marine invertebrates the guidance is done by chemotaxis. In the case of mammals, it appears to be done by chemotaxis, thermotaxis and rheotaxis.
Sperm chemotaxis is a form of sperm guidance, in which sperm cells (spermatozoa) follow a concentration gradient of a chemoattractant secreted from the oocyte and thereby reach the oocyte.
Virus quantification involves counting the number of viruses in a specific volume to determine the virus concentration. It is used in both research and development (R&D) in commercial and academic laboratories as well as production situations where the quantity of virus at various steps is an important variable. For example, the production of viral vaccines, recombinant proteins using viral vectors and viral antigens all require virus quantification to continually adapt and monitor the process in order to optimize production yields and respond to ever changing demands and applications. Examples of specific instances where known viruses need to be quantified include clone screening, multiplicity of infection (MOI) optimization and adaptation of methods to cell culture. This page discusses various techniques currently used to quantify viruses in liquid samples. These methods are separated into two categories, traditional vs. modern methods. Traditional methods are industry-standard methods that have been used for decades but are generally slow and labor-intensive. Modern methods are relatively new commercially available products and kits that greatly reduce quantification time. This is not meant to be an exhaustive review of all potential methods, but rather a representative cross-section of traditional methods and new, commercially available methods. While other published methods may exist for virus quantification, non-commercial methods are not discussed here.
An organ-on-a-chip (OOC) is a multi-channel 3-D microfluidic cell culture, integrated circuit (chip) that simulates the activities, mechanics and physiological response of an entire organ or an organ system, a type of artificial organ. It constitutes the subject matter of significant biomedical engineering research, more precisely in bio-MEMS. The convergence of labs-on-chips (LOCs) and cell biology has permitted the study of human physiology in an organ-specific context, introducing a novel model of in vitro multicellular human organisms. One day, they will perhaps abolish the need for animals in drug development and toxin testing.
Virtual colony count (VCC) is a kinetic, 96-well microbiological assay originally developed to measure the activity of defensins. It has since been applied to other antimicrobial peptides including LL-37. It utilizes a method of enumerating bacteria called quantitative growth kinetics, which compares the time taken for a bacterial batch culture to reach a threshold optical density with that of a series of calibration curves. The name VCC has also been used to describe the application of quantitative growth kinetics to enumerate bacteria in cell culture infection models. Antimicrobial susceptibility testing (AST) can be done on 96-well plates by diluting the antimicrobial agent at varying concentrations in broth inoculated with bacteria and measuring the minimum inhibitory concentration that results in no growth. However, these methods cannot be used to study some membrane-active antimicrobial peptides, which are inhibited by the broth itself. The virtual colony count procedure takes advantage of this fact by first exposing bacterial cells to the active antimicrobial agent in a low-salt buffer for two hours, then simultaneously inhibiting antimicrobial activity and inducing exponential growth by adding broth. The growth kinetics of surviving cells can then be monitored using a temperature-controlled plate reader. The time taken for each growth curve to reach a threshold change in optical density is then converted into virtual survival values, which serve as a measure of antimicrobial activity.
A ligand binding assay (LBA) is an assay, or an analytic procedure, which relies on the binding of ligand molecules to receptors, antibodies or other macromolecules. A detection method is used to determine the presence and extent of the ligand-receptor complexes formed, and this is usually determined electrochemically or through a fluorescence detection method. This type of analytic test can be used to test for the presence of target molecules in a sample that are known to bind to the receptor.
Collective motion is defined as the spontaneous emergence of ordered movement in a system consisting of many self-propelled agents. It can be observed in everyday life, for example in flocks of birds, schools of fish, herds of animals and also in crowds and car traffic. It also appears at the microscopic level: in colonies of bacteria, motility assays and artificial self-propelled particles. The scientific community is trying to understand the universality of this phenomenon. In particular it is intensively investigated in statistical physics and in the field of active matter. Experiments on animals, biological and synthesized self-propelled particles, simulations and theories are conducted in parallel to study these phenomena. One of the most famous models that describes such behavior is the Vicsek model introduced by Tamás Vicsek et al. in 1995.
A wound healing assay is a laboratory technique used to study cell migration and cell–cell interaction. This is also called a scratch assay because it is done by making a scratch on a cell monolayer and capturing images at regular intervals by time lapse microscope.
Microfluidic cell culture integrates knowledge from biology, biochemistry, engineering, and physics to develop devices and techniques for culturing, maintaining, analyzing, and experimenting with cells at the microscale. It merges microfluidics, a set of technologies used for the manipulation of small fluid volumes within artificially fabricated microsystems, and cell culture, which involves the maintenance and growth of cells in a controlled laboratory environment. Microfluidics has been used for cell biology studies as the dimensions of the microfluidic channels are well suited for the physical scale of cells. For example, eukaryotic cells have linear dimensions between 10-100 μm which falls within the range of microfluidic dimensions. A key component of microfluidic cell culture is being able to mimic the cell microenvironment which includes soluble factors that regulate cell structure, function, behavior, and growth. Another important component for the devices is the ability to produce stable gradients that are present in vivo as these gradients play a significant role in understanding chemotactic, durotactic, and haptotactic effects on cells.
The enzyme-linked immune absorbent spot (ELISpot) is a type of assay that focuses on quantitatively measuring the frequency of cytokine secretion for a single cell. The ELISpot Assay is also a form of immunostaining since it is classified as a technique that uses antibodies to detect a protein analyte, with the word analyte referring to any biological or chemical substance being identified or measured.
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