Attune™ Xenith™ Flow Cytometer

Yes, the Attune Xenith Flow Cytometer has built in FSC and SSC channels off the violet laser so that no-lyse/no-wash assays, which exploit the difference in violet light-scattering properties between red blood cells and leukocytes, can be easily run without the need for any changes in filters or need for purchase of additional accessory kits. This integrated capability helps make the process straightforward and efficient.

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Choosing between spectral and conventional flow cytometry depends on your experimental needs and the complexity of your panels. Spectral flow cytometry offers significant advantages for high-parameter detection, improved data resolution, flexibility, simplified panel design, and the ability to handle complex samples. It is also beneficial for sample types with multiple auto fluorescent populations to be explored and for panels where a user may not be familiar with the best peak channel to choose. Conventional flow cytometry remains a robust and effective option for simpler applications and lower-parameter analyses. Conventional compensation can be beneficial for panels of smaller size and/or limited single-channel overlap. It can also be beneficial for panels that include very bright dyes such as functional dyes which often require individual detector adjustments (adjusting the full spectral signature to keep these dyes on scale in bright samples may lead to confusing spectral signatures). By considering these factors, you can select the flow cytometry method that best aligns with your research goals and requirements.

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Both beads and cells can be used as controls in spectral unmixing, and the choice between them depends on the specific requirements and goals of the experiment. Beads offer consistency, reproducibility, and ease of use, making them ideal for calibration and routine quality control. Cells, on the other hand, provide biological relevance and allow for the characterization of complex spectra and autofluorescence, which can be crucial for certain applications. Therefore, the decision to use beads or cells should be based on the specific needs of the spectral unmixing process and the experimental context. Cells are a good choice if you have plenty of sample, the antibody in use will stain a large and distinct positive population, and there will be an internal negative population. Beads are a good choice if your sample is limited, the antibody would only stain a very small subset of cells, or the population of cells that would stain would not be distinct. Beads are also only a good choice if the spectral characteristics of the fluorophore are maintained on beads. We recommend that when establishing a panel, a full set of single-stain controls of both beads and cells be prepared. After recording the controls, the user should check the signature of the beads vs cells and decide on the best choice for each fluorophore. For example, if the spectral signatures are different on beads, then cells will be needed; and if cells are brighter than beads, then the cells would need to be used for the controls. It is likely that a mixture of cells and beads for controls will be the optimal choice.

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The Attune Xenith Flow Cytometer performs spectral unmixing by capturing the emission spectrum of each fluorophore across multiple detectors. It uses advanced spectral unmixing algorithms to accurately deconvolute overlapping spectra, helping ensure precise identification and quantification of each fluorophore. Specifically, the Attune Xenith Flow Cytometer employs the Ordinary Least Squares (OLS) algorithm for spectral unmixing, which is a widely used method in spectral systems. This approach allows for accurate separation of signals, even when spectra overlap significantly, helping ensure high-quality data for complex multi-parameter analyses.

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The Attune Xenith Flow Cytometer uses Photomultiplier Tubes (PMTs) instead of Avalanche Photodiodes (APDs) due to several critical advantages. PMTs offer high sensitivity, allowing them to detect very low levels of light, which is essential for precise and accurate measurements in flow cytometry. They also provide a wide dynamic range, capable of measuring a broad range of signal intensities from very dim to very bright, thus enabling detailed quantification across various sample conditions. Additionally, PMTs have a fast response time, quickly reacting to changes in light intensity, which is crucial for the rapid data acquisition required in flow cytometry. Their spectral flexibility allows the use of various optical filters to detect different wavelengths of light, enhancing their versatility in multi-parameter analysis. Moreover, PMTs are highly reliable and compatible with advanced optical systems, helping ensure consistent and high-quality data collection. In contrast, while some APDs may offer higher quantum efficiency, they suffer from a higher noise floor and limited linearity for bright signals, which negatively impacts the flexibility and accuracy needed to run both compensation and spectral panels on the same instrument. These attributes make PMTs an excellent choice for achieving precise, accurate, and high-quality data in both spectral and conventional flow cytometry applications.

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