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High-parameter immune profiling is crucial in translational and clinical research to quantify changes in immune cell populations over time. CyTOF® mass cytometry is a high-plex, single-cell analysis platform that uses isotopically pure metal-labeled antibodies. The major advantage of CyTOF is its ability to resolve 40-plus markers in a single panel without compensation, making mass cytometry ideal for routine immunophenotyping. The autosampler module of CyTOF XT™ provides significant time savings by allowing automated sample acquisition. Tubes of pelleted stained samples are loaded into the Autosampler carousel, and the samples are resuspended with EQ™ Calibration Beads for acquisition. User input is only required during instrument startup, tuning, and batch setup. The added automation of CyTOF XT provides a streamlined workflow for suspension mass cytometry. Testing was performed to ensure that the data obtained on CyTOF XT was comparable to manual acquisition systems. The performance of CyTOF XT was tested in parallel with its predecessor, Helios™. Several workflows and applications for suspension mass cytometry including sample barcoding with the Cell-ID™ 20-Plex Pd Barcoding Kit, and surface, cytoplasmic, and nuclear staining and phosphostaining were evaluated on human PBMC. Manual gating analysis was performed to assess population frequencies and median intensities for each marker. Resolution index was calculated to assess how well positive and negative populations separated from each other. There was no significant difference between population frequencies analyzed between the two CyTOF systems. Moreover, samples acquired on CyTOF XT, on average, resulted in greater signal resolution between positive and negative populations compared to Helios. The Maxpar Direct Immune Profiling System was also compared on CyTOF XT and Helios using human whole blood and PBMC. The Maxpar® Direct™ Immune Profiling Assay™ and Maxpar Pathsetter™ software were developed as a sample-to-answer system for human immune profiling using CyTOF. The Maxpar Direct Immune Profiling Assay includes an optimized panel of 30 unique markers in a dry, single-tube format. Maxpar Pathsetter is an automated software used to report population statistics, stain assessments, and relevant data plots for the panel. The automated staining assessment in Maxpar Pathsetter was compared between files acquired on CyTOF XT and Helios. Comparable population frequencies were obtained between the two acquisition systems, and improved staining assessment was observed on CyTOF XT. Overall, these studies demonstrate that CyTOF XT generates better signal resolution as compared to Helios. The automated acquisition of CyTOF XT enables researchers to streamline immunophenotyping of human samples while accurately and reproducibly monitoring changes in immune cell subsets. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Thiru Selvanantham, Stephen K.H. Li, Nick Zabinyakov, Alexandre Bouzekri, Raymond Jong, Michael Sullivan, Alexander Laboda, Daniel Majonis, Christina Loh. A streamlined and automated approach to high-content cytometric immunophenotyping with CyTOF XT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3908.