Invitrogen™ Attune™ NxT Flow Cytometer

Platelets are non-nucleated cellular fragments that circulate in the peripheral blood. They derive from megajaryocytes and are critical for maintaining hemostasis. This application note demonstrates the use of the Attune NxT Flow Cytometer, along with the Attune NxT No-Wash and No-Lyse Filter Kit for violet laser SSC detection as a robust assay for the detection of platelets in white blood without sample manipulation.

The Invitrogen™ eBioscience™ Super Bright polymer dyes represent a suite of bright fluorophores excited by the violet laser (405 nm). Optimized for use in flow cytometry, the Super Bright dyes allow for expanded use of violet laser excitation and promote streamlined multicolor panel design. This scientific poster describes the use of these dyes and the Invitrogen™ Attune™ NxT Flow Cytometer to characterise human T-cell lymphocytes.

Immunophenotyping is the analysis of heterogeneous populations of cells within mouse blood, splenocytes, and thymocytes for the purpose of identifying the presence and proportions of various cell populations. Flow cytometry is the method of choice for identifying cells within complex populations, as it allows for multiparameter analysis of thousands to millions of cells in a short time. This application note describes the use of the Attune NxT Flow Cytometer for 4-laser and 10-color immunophenotyping analysis of stained mouse splenocytes using a fixation and permeabilization protocol.

Immunophenotyping is the analysis of heterogeneous populations of cells within human blood for the purpose of identifying the presence and proportions of various cell populations. Multiparameter flow cytometry is a widely-used method for identifying cell subsets within complex cellular systems. This application note describes the use of the Attune NxT Flow Cytometer for 4-laser and 10-color immunophenotyping analysis of stained human whole blood using a stain-and-lyse protocol.

Fluorescent proteins are a valuable tool for researchers. A world of possibilities has been opened through their discovery and incorporation into biological research. The ability to monitor both live and dead manipulated cells allows researchers to better understand the mechanisms that regulate cellular processes. However, in order to utilize this, it is necessary to be able to analyze these processes on an individual cellular basis. Fortunately, with the increasing abilities of flow cytometers like the Attune NxT Flow Cytometer with up to 16 parameters of detection and clogresistant design, researchers can now explore what was overly difficult or simply impossible only a few years ago.

Compensation is one of the most critical, and yet poorly understood, topics in flow cytometry. Just as setting the proper voltage and knowing the range of wavelengths where fluorophores excite and emit, compensation is necessary for successful flow cytometry. Compensation is required for flow cytometry experiments because of the physics of fluorescence and the construction of flow cytometers. A fluorophore is excited and emits a photon in a range of wavelengths. Some of these photons spill into a second detector, causing single-stained samples to appear double positive. A mathematical correction is applied to flow cytometry data to address photon spillover— this is compensation. This application note describes the use of the Invitrogen™ Attune™ NxT Flow Cytometer for 6-color immunophenotyping analysis of stained human whole blood using a no-lyse, no-wash protocol.

This application note describes the use of the Attune NxT Flow Cytometer for 3-color immunophenotyping analysis of stained mouse splenocytes using the Foxp3 Transcription Factor Staining Buffer Kit, a fixation and permeabilization kit designed to work with transcription factors, like Foxp3, and other intracellular markers. Regulatory T cells come in many forms, with the most understood being those that express CD4, CD25, and Foxp3 (CD4+ CD25+ regulatory T cells). CD4+ CD25+ regulatory T cells are a specialized subpopulation of T cells that act to maintain homeostasis within the immune system by suppressing the immune response of other cells. This is an important “self-check” built into the immune system to prevent excessive reactions.

Immunophenotyping is the analysis of heterogeneous populations of cells within human blood for the purpose of identifying the presence and proportions of various cell populations. This application note describes the use of the Attune NxT Flow Cytometer with 4 lasers and 13-color immunophenotyping analysis of stained human whole blood using a stain/lyse protocol. Lymphocyte, monocyte and granulocyte populations were distinguished with forward scatter (FSC) and side scatter (SSC); and monocyte, T cell, B cell and NK populations were identified using fluorescent antibodies against surface antigens specific for the different immunological populations.

Standard methods for isolating and detecting leukocytes (white blood cells) in human whole blood are time-consuming and often involve significant manipulation and enrichment prior to analysis. In human whole blood, red blood cells outnumber white blood cells ~1,000-fold. This creates two hurdles in attempting to analyze whole blood samples without manipulation or enrichment: 1) collection of a sufficient number of white blood cell events for statistically meaningful data, and 2) differentiation of white blood cells from red blood cells given the high probability of coincident red blood cell events, which is clear when observing a conventional forward and side scatter profile of whole blood. This application note outlines a strategy for the detection of white blood cells within whole blood using the Attune NxT Flow Cytometer.

This application note presents a few major obstacles in rare-cell detection, specific strategies to address them, and examples of successful rare-cell analysis by flow cytometry. It is also demonstrated that acoustic focusing cytometry can dramatically increase sample acquisition rates compared with conventional flow cytometry, enabling a larger number of rare cells to be analyzed in a single experiment.

In this video, Greg Kaduchak, principal scientist at Thermo Fisher Scientific, discusses the importance of overcoming flow cytometry setbacks, and reveals how to effectively deliver high-resolution brightfield images. Kaduchak also explores how the Attune CytPix Flow Cytometer from Thermo Fisher Scientific could be the solution for you, highlighting the range of customer feedback received so far.

Cancer stem cells (CSCs) are rare tumor cells which exhibit high levels of drug resistance. Steve McClellan, B.S, M.T., SCYM (ASCP), the Flow Cytometer and Imaging Core Lab Manager at the Mitchell Cancer Institute, describes his team’s research into how exosomes enable CSCs to communicate with other cells. Steve also explains how they have developed a new method for analyzing exosomes using the Invitrogen™ Attune™ NxT Flow Cytometer and why he thinks it will be a breakthrough in the field.

Watch this video to hear how the Invitrogen™ Attune™ NxT acoustic focusing flow cytometer is key to a cutting-edge research project investigating antibiotic resistance. The Thermo Fisher Scientific flow cytometer enables scientists at the University of Western Australia to quickly and accurately profile the effect of drugs on individual bacterial cells directly from clinical samples.

In this video, hear Jace Akerlund describe the Invitrogen™ Attune™ NxT Flow Cytometer which is enabling rapid analysis of samples. Built with modularity in mind, the Attune can function with between one and four lasers, providing a variety of colored systems and allowing for add-ons to be purchased as budget allows.

Watch this video to find out how the Autónoma University of Madrid is using the Invitrogen™ Attune™ NxT Flow Cytometer for acoustic-based flow cytometry. This technology enables reliable and efficient identification and isolation of cells from tumors and liquid biopsies.

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Current applications of flow cytometry in the clinical laboratory

Dr Charles Prussak will receive the grant to help further his research on immune-cell therapy for cancer treatment

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