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

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.