MACSQuant® Analyzer 10 Flow Cytometer

The determination of cell viability in any cell culture is a crucial analytical measurement for any study. This includeshigh-throughput cytotoxicity screens of large libraries of compounds and drug discovery research, and studies in all laboratories interested in validating their cell cultures upstream or downstream of any given application.

Neural stem cells (NSCs) in the adult subventricular zone (SVZ) and the dentate gyrus have the capacity to self-renew and generate new neural cells throughout their lifetime. Their ability to react to brain injury by generating new neurons makes them a valuable cell source for endogenous repair in the adult brain. NSCs are a very rare and sensitive cell population, and a complex marker combination is necessary to distinguish them from other cells. Therefore, their purification still poses a great challenge.

Successful cell sorting via flow cytometry highly depends on the collection of viable cells. It is well established that high pressure, decompression, and shear forces negatively affect cell viability and functionality during conventional droplet sorting. Therefore, to mitigate the risk of cell damage, instrument settings, including nozzle size and pressure, have to be carefully chosen by experienced operators, who are capable of adjusting the settings accordingly. Sterility is another concern when working with traditional cell sorters that often operate in an uncontrolled, open-air surrounding. Samples are prone to contamination from both internal sources (sheath fluid, sampling fluidics, etc.) and external sources (airborne contaminants).

With adoptive T cell therapies (ACTs) becoming more prevalent, the need for easy and effective isolation of antigen-specific T cells in a clinical setting is growing. The MACSQuant® Tyto™ is a benchtop cell sorter that is fully closed, sterile, and easy to use. At the heart of the system is the disposable, single-use cartridge, which allows for completely aseptic sorting conditions with no chance of cross-contamination between samples. Here we demonstrate the MACSQuant Tyto’s capacity to sort rare antigen-specific cytotoxic T cells out of fresh peripheral blood mononuclear cells (PBMCs). As a model we used PBMCs from a CMV-seropositive donor. The PBMCs contained less than 0.1% of CMV-specific T cells. CMV is a member of the herpes virus group and has a prevalence of 50–96% in human adults. Once infected, the virus persists in the body. The infection is asymptomatic in healthy individuals, but in immunocompromised patients CMV can cause severe diseases.

Cell counting is an often overlooked, yet critical basis for experiments and production processes. Methodology for cell counting spans multiple technologies that include fluidic-based systems or immobilized slide-based techniques. These methods range from manual to fully automated counting, depending on the device. While there are numerous options available, consistency and reproducibility often fall short. Especially manual counting techniques can be extremely variable with coefficients of variation (CV) as high as 27%.

Fluorescence resonance energy transfer (FRET) is a mechanism describing an energy transfer between two fluorescent molecules. The efficiency of this energy transfer is inversely proportional to the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance. Measurements of FRET efficiency can be used to determine if two fluorochromes are within a certain distance of each other. Therefore FRET can be used to detect molecular interactions in a number of systems and has hence often been applied in various assays in biology and chemistry.

Infection-related mortality is a considerable clinical challenge in immunocompromised individuals. Pathogenspecific T cells are crucial mediators of immune protection as shown for example by adoptive transfer of antigenspecific T cells. However, for both predicting or diagnosing infectious complications as well as for the development of effective therapies it is crucial to have reliable methods to phenotypically and functionally characterize the antigenspecific T cells.

The activation of natural killer T (NKT) cells – a regulatory T cell subset – during the immune response is thought to be reliant on endogenous glycolipid display. Many potential candidates for these NKT cell self- antigens have been proposed, including β-linked glycosylceramides. However, determining their actual identity has been difficult due to the low sensitivity of the chemical methods that could not rule out low-level α-anomer contamination of natural or synthetic β-anomer preparations.

Follicular T helper cells (Tfh) play a crucial role in supporting germinal center induction and responses by providing B cell help needed for affinity maturation and generation of B cell memory. Circulating (cTfh) have moved to the center stage of immunological research since it becomes increasingly apparent that these cells are providing insights into the immune responses occurring in the germinal centers of lymphoid organs. Typically, these lymphatic tissues are not accessible when studying human immune responses, thus making cTfh an attractive target for surrogate markers of immunity.

Preclinical safety assessments often include the in vitro micronucleus assay in order to evaluate chemicals' potential to cause DNA damage. This particular assay provides information on chromosomal damage in cultured cells by detecting the formation of small membrane-bound DNA fragments (micronuclei) in the cytoplasm of interphase cells. Traditionally, the in vitro micronucleus assay requires a microscopic analysis of the treated cells which precludes its use as a high-throughput screening tool.

To develop a representative of the corticospinal system, Dr. Erna A. van Niekerk, University of California San Diego (UCSD), presents a new method to isolate and culture viable neurons from mice at postnatal day 12 and 20. This cellular representation of corticospinal neurons, she hopes, will advance drug screening for spinal cord injury, stroke, ALS and other disorders.

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