Modern flow cytometry experiments routinely measure 18 or more fluorescent markers across many samples, patients, and tissues. As these experiments' complexity increases, manual gating becomes unacceptably inefficient and can introduce operator-to-operator variation. Computational cytometry algorithms such as unsupervised clustering, automated gating, and dimensionality reduction can increase the speed and reliability of these analyses, but using them requires time and expertise that many biomedical scientists lack. Cytoflow was built to bridge this gap. Cytoflow is open source, user-friendly point-and-click software written in Python that allows non-programmers to apply modern computational flow cytometry methods to their data sets. Additionally, Cytoflow's modules can be used directly in a Python script or a JupyterLab notebook. Finally, extending Cytoflow with new modules that support future applications is straightforward, and feedback from an active user community continues to guide ongoing development. As a result, Cytoflow can save an experimenter time and improve the reliability and reproducibility of their analysis. Cytoflow is available to download at https://cytoflow.github.io. Source code is hosted at https://github.com/cytoflow/cytoflow, and documentation is available at https://cytoflow.readthedocs.io.
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