T Lymphocyte Immunophenotyping: 14-Color Flow Cytometry Panel Design Using the Attune NxT Flow Cytometer and Super Bright Fluorescent Dyes

Abstract

Abstract 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. Due to their inherent brightness, detection of cell populations with low abundance targets is possible. A 14-color panel used to characterize human T-cell lymphocytes is presented, with information on differentiation profiles, activation or exhaustion status, and co-stimulation activity. Both cryopreserved human peripheral blood mononuclear cells (PBMC) and fresh blood collected from a normal donor was used. Acquisition and analysis was performed using the Attune™ NxT Flow Cytometer four laser system, which has the ability to detect multiple violet-excited fluorophores. The results show well characterized cell populations, with expected changes between unstimulated and stimulated PBMC ex vivo. Correction for spectral overlap was performed using standard instrument auto-compensation procedures. The unique acoustic focusing technology of Attune NxT Flow Cytometer enables rapid and accurate detection with sensitivity at high flow rates without compromise of data quality, as demonstrated with this 14-color data set. The Attune NxT cytometer is designed to accommodate a wide variety of experimental conditions and combined with the Super Bright dyes provides expanded choice for panel design. For research use only. Not for use in diagnostic procedures.