No-lyse, No-wash Protocol for 6-color Immunophenotyping Analysis of Stained Human Whole Blood without Compensation using the Attune NxT Flow Cytometer

No-lyse, No-wash Protocol for 6-color Immunophenotyping Analysis of Stained Human Whole Blood without Compensation using the Attune NxT Flow Cytometer

18 Feb 2016

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.

Invitrogen™ Attune™ NxT Flow Cytometer

Thermo Fisher Scientific

The Invitrogen™ Attune™ NxT flow cytometer can be designed to accommodate the most common fluorophores and fluorescent proteins used in flow cytometry to match the panels you are currently running. Multiple fluorescent proteins can be detected with an optional 561 nm laser. Whether you configure your system now or upgrade later, the Attune NxT flow cytometer can grow with you and your research needs. The Attune NxT flow cytometer, designed using acoustic-assisted hydrodynamic focusing, achieves sample-throughput rates of of 12.5 µL/min–1 mL/min or 12.5–1,000 µL/min—up to 10 times faster than traditional hydrodynamic focusing systems and data acquisition speeds of 35,000 events/sec. Acoustic focusing allows significantly higher sample rates, which simplifies sample preparation, reduces protocol times, and minimizes cell loss. This feature is useful for no wash, no lyse immunophenotyping protocols using whole blood and for samples that are inherently low in concentration, including cerebrospinal fluid and stem cell preparations. Your next result relies on the ability of your experimental design and your instrument to deliver more—more data, more detail, and more throughput. The Attune NxT flow cytometer was designed to meet the demands of more complex experiments, offering reliable data, day after day, in a grow-with-you system.   Features & Benefits: Modular design—Multiple configurations available—field upgradable. Save time—10X faster speeds with no loss in data quality. Simplified sample prep—No wash, no lyse options, non-clogging fluidics. Enables unique applications—Complex protocols on a broad range of cell types and samples.

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No-lyse, No-wash Protocol for 6-color Immunophenotyping Analysis of Stained Human Whole Blood without Compensation using the Attune NxT Flow Cytometer